WO1998051802A1 - Method for the stabilization of proteins and the thermostabilized alcohol dehydrogenases produced thereby - Google Patents

Method for the stabilization of proteins and the thermostabilized alcohol dehydrogenases produced thereby Download PDF

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WO1998051802A1
WO1998051802A1 PCT/US1998/009627 US9809627W WO9851802A1 WO 1998051802 A1 WO1998051802 A1 WO 1998051802A1 US 9809627 W US9809627 W US 9809627W WO 9851802 A1 WO9851802 A1 WO 9851802A1
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val
lys
ala
ser
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PCT/US1998/009627
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French (fr)
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David C. Demirjian
Igor A. Brikun
Malcolm J. Casadaban
Veronika Vonstein
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Thermogen, Inc.
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Priority to CA002290074A priority Critical patent/CA2290074A1/en
Priority to AU73808/98A priority patent/AU7380898A/en
Publication of WO1998051802A1 publication Critical patent/WO1998051802A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • the present invention generally relates to a method for the directed evolution of proteins.
  • the method is directed to stabilization of proteins such as dehydrogenases, and particularly is directed to a method for improving the thermostability of dehydrogenases such as alcohol dehydrogenases.
  • the present invention also relates to thermostabilized alcohol dehydrogenases produced according to this method.
  • Biocatalysts are enzymes which can specifically and efficiently expedite chemical reactions such as the synthesis of chemical compounds and biopolymers (Dixon et al . , Enzymes (Academic Press, New York: 1979)) . Biocatalysts are the key players in a number of important industrial synthetic and degradative applications including, but not limited to, the following:
  • Biodegradation Applications are employed as enzymatic degradation agents for environmental pollutants such as PCBs, chlorinated hydrocarbons, RDX, halogenated organic compounds, TNT, and other byproducts of industrial production that present significant health risks.
  • environmental pollutants such as PCBs, chlorinated hydrocarbons, RDX, halogenated organic compounds, TNT, and other byproducts of industrial production that present significant health risks.
  • alcohol dehydrogenases are enzymes that command formal, reversible, two-electron chemistry in which alcohols are oxidized to the corresponding ketones.
  • ketones can be reduced to the respective alcohols via a stereospecific delivery of a hydride equivalent catalyzed by the enzyme coupled to a bound cofactor such as NADH or NADPH (Lemiere, "Alcohol Dehydrogenase Catalyzed Oxidoreduction Reactions in Organic Chemistry", I_n Enzymes as Catalysts in Organic Synthesis, Schneider et al . , Eds. (1986) p.
  • This system thus provides a mild, extremely sensitive route to chiral compounds, without contamination from undesired, competing reactions .
  • Such chiral compounds can be used, especially by the pharmaceutical industry, for the preparation of chiral therapeutics, and for effectively generating a wide variety of compounds having the capacity for industrial scale-up (Seebach et al . , Org . Synth . , 63, l-_ (1984); Bradshaw et al . , J. Org. Chem. , 57, 1532(1992); Hummel, Biotechnol . Lett . , 12, 403(1990)).
  • dehydrogenases show promise for commercial application in the preparation of unusual amino acids and ⁇ - hydroxyketones , and in the resolution of racemic alcohols (Benoiton et al . , J. Am. Chem. Soc ⁇ , 79, 6192 (1957);
  • HLADH horse liver alcohol dehydrogenase
  • biocatalyst such as HLADH
  • HLADH enzyme-catalyst
  • one of the greatest challenges associated with biocatalyst implementation is that of overcoming an overall intrinsic instability that results in a requirement for special preparative approaches and handling conditions. Many methods have been used m an attempt to stabilize certain proteins.
  • Rational protein engineering has allowed the redesign of proteins with altered properties such as enhanced stability, shifted pH optima, and different substrate specificities (see, e.g., Bryan et al . , Proteins, 1 ⁇ , 326-334 (1986); Pantoliano et al . , Biochemistry, 26, 2077-82 (1987); Carter et al . , Science, 237, 394-399 (1987); Wells et al . , "Designing substrate specificity by protein engineering of electrostatic interactions", , 84 , 1219-1223 (1987) ;
  • the present invention seeks to overcome some of the aforesaid problems of enzyme design.
  • Such a method of stabilizing dehydrogenases (particularly HLADH) would present a major advancement in the field since it would extend the shelf life, longevity, and active temperature range of these enzymes.
  • the present invention provides, inter alia , a method for the stabilization of a protein (particularly for the stabilization of an alcohol dehydrogenase such as horse liver alcohol dehydrogenase (HLADH) , general enrichment/selection means that can be employed in Escherichia and Thermus to select for cells having altered levels of alcohol dehydrogenase activity as compared to a wild-type cell, thermostabilized HLADH proteins and nucleic acid sequences encoding same, as well as plasmids and hosts cells comprising the nucleic acid sequences .
  • an alcohol dehydrogenase such as horse liver alcohol dehydrogenase (HLADH)
  • general enrichment/selection means that can be employed in Escherichia and Thermus to select for cells having altered levels of alcohol dehydrogenase activity as compared to a wild-type cell
  • thermostabilized HLADH proteins and nucleic acid sequences encoding same as well as plasmids and hosts cells comprising the
  • FIGURES Figure 1 is a diagram that generally depicts the approach of the present invention for the accelerated evolution of enzymes.
  • a pool of mutants of the particular gene is obtained by means such as spontaneous, directed, chemical, or PCR-mediated mutagenesis.
  • the mutants of interest i.e., having the particular stabilized feature
  • Figure 2 is a digitized image of results of a filter assay for alcohol dehydrogenase activity which demonstrates that wild-type HLADH is rapidly inactivated at 75 °C: no heat treatment (A) ; 5 minutes of heat treatment at 75 °C (B) ; 10 minutes of heat treatment at 75 °C (C) ; 15 minutes of heat treatment at 75 °C (D) ; 20 minutes of heat treatment at 75 °C (E) ; and 50 minutes of heat treatment at 75 °C (F) .
  • Figure 3 is a partial restriction map of the plasmid pTG450 which contains the adh gene from plasmid pBPP cloned into a pTG100kan tr2 Thermus shuttle vector.
  • Figure 4 is a bar chart that depicts the increased thermostability of HLADH mutants produced according to the invention at 70°C. Cells containing pGEM-T (i.e., having no HLADH gene) did not show any HLADH activity.
  • Figure 5 is the sequence of adh gene [SEQ ID NO:l] that encodes the HLADH protein [SEQ ID NO: 2] , with the location of certain mutations produced according to the invention identified as the boxed regions.
  • the present invention provides, among other things, a method for stabilizing a certain feature of a protein (e.g., stability at a certain temperature, stability in the presence of certain reagents, etc.) .
  • the method of the invention provides a method for thermostabilizing a protein.
  • the invention preferably provides a method of obtaining nonnative protein having a thermostability that is increased over that of the native version of said protein, as further described herein.
  • a "native" protein is the protein as it generally is found in nature.
  • a “nonnative” protein differs from the native protein in that it has been modified by human intervention, i.e., at either the level of the protein or its encoding DNA (e.g., by recombinant means to directly alter the genome; by unique selection and forced mutation; by random mutagenesis) .
  • a "protein” desirably can be either an entire protein, or a portion of a protein (e.g., as where a chimeric nonnative protein results from either transcriptional or translational gene fusion) .
  • a "nonnative protein” in some applications may be a peptide (i.e., an incomplete protein) , as where the peptide is chemically synthesized or, where a gene's coding sequence is transcribed or translated in vi tro or, is produced by chemical processing of a complete protein.
  • a preferred protein for stabilization, particularly thermostabilization according to the invention is a dehydrogenase, particularly an alcohol dehydrogenase, and especially horse liver alcohol dehydrogenase (e.g., as obtained from plasmid pBPP, and/or as set forth in SEQ ID NO: 2) .
  • this protein does not initiate with methionine (Met) .
  • other varients of horse liver alcohol dehydrogenase produced by in vi tro synthetic reactions by means of chemical synthesis or, in other hosts (e.g., an eukaryotic host or other prokaryotic host cell) may possess a methionine residue in the first position of the protein.
  • a protein according to the invention optionally can be another type of dehydrogenase, e.g., another type of NAD+ (P) -linked dehydrogenase including, but not limited to, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase (NADP+) , hydroxylacyl CoA dehydrogenase, glyceraldehyde 3 -phosphate dehydrogenase, and glucose 6- phosphate dehydrogenase (NADP+) .
  • NAD+ NAD+
  • the method can be employed to thermostabilize a horse liver alcohol dehydrogenase.
  • This method generally is depicted in Figure 1.
  • the method comprises: (a) obtaining in a vector a gene that encodes the native protein; (b) mutating the vector at more than one position in the gene to produce a vector library of cells comprising mutated versions of the gene;
  • gene that encodes said protein can comprise a recombinant or nonrecombinant sequence, i.e., a sequence that is present as found in nature (i.e., encodes a native amino acid sequence) or, has been modified, for instance by the introduction of mutations (e.g., point mutations, insertions, deletions, or rearrangements) to comprise a nonnative amino acid sequence or, can be a mixture of native and nonnative amino acid sequences.
  • a recombinant gene may conjoin coding sequences (either in entirety or in part) with regulatory sequences (e.g., transcription initiation, transcription termination, translational start or stop sites, protein secretion sequences, and the like) which are not typically conjoined in nature.
  • regulatory sequences e.g., transcription initiation, transcription termination, translational start or stop sites, protein secretion sequences, and the like. This can allow the production of a protein in a host in which it normally is not produced (e.g., production of a eukaryotic protein in a prokaryotic cell) .
  • the recombinant gene (which can derive, in entirety or part, from any prokaryotic, eukaryotic, bacteriophage , or viral source) is capable of being transcribed and translated in a prokaryotic cell, particularly, a cell comprising a member of the genuses Escherichi or Thermus .
  • a host cell in the context of the present invention i.e., which can be employed in a method of stabilizing proteins
  • a cell employed in the method of stabilizing (particularly thermostabilizing) proteins according to the invention is a thermophile or hyperthermophile .
  • a cell is a member of the genus Thermus , and desirably is of the species Thermus flavus, Thermus aqua ticus, Thermus thermophilus, or Thermus sp .
  • a cell is either an Escherichia coli cell or a Thermus aquaticus cell.
  • the vector in which the gene of interest is subcloned can be any vector appropriate for delivery of a gene to a cell.
  • the vector can be a plasmid, bacteriophage, virus, phagemid, cointegrate of one or more vector species, etc.
  • a vector is one that can be employed for gene expression in a prokaryotic cell such as a Thermus or Eshcerichia cell. It also is preferable that a vector have an ability to shuttle between different cells, e.g., between a Thermus and an Eschericia cell.
  • One such vector that can be employed in the context of the invention is the vector pTG450.
  • the preferred method of the invention calls for mutating a vector containing the gene encoding the protein to be stabilized.
  • Any method of mutagenesis such as is known to those skilled in the art and particularly as is described in the following Examples, can be employed in the method of the invention for generating a mutated gene.
  • a PCR-based (error prone) approach is employed for mutagenesis.
  • other mutagens e.g., chemical mutagens such as hydroxylamine
  • the vector is mutated at more than one position in the gene of interest. This can be assessed by means known in the art and as described in the Examples.
  • Such mutagenesis in more than one position in the gene will result in a "vector library" comprising mutated versions of a gene, particularly of a horse liver alcohol dehydrogenase gene, which are present in the library mixture.
  • the vector library can be introduced en masse into cells (e.g., by transformation) . Since the vectors and the cells employed for these methods are selected to be compatible, and the gene is engineered (e.g., as described below) to contain or to be flanked by any sequences necessary for its expression, it is expected that such introduction will result in the transcription and ensuing translation of the introduced gene. Moreover, such en masse introduction will result in the generation of a cell library comprising a mixture of cells transformed with plasmids having differing mutated genes.
  • the cells preferably are screened under conditions that allow identification of a cell comprising a mutated version of the gene of interest that encodes a nonnative protein having a protein that is stabilized (e.g., thermostabilized) over that of the wild-type (i.e., native) versions of the protein.
  • a variety of selection means can be employed in accordance with the method of the present invention and, in particular, the selection means identified in the Examples which follow can be employed. Of course, one of ordinary skill in the art could modify these methods such that they are adapted for a particular host cell and/or a particular protein of interest.
  • screening conditions are employed that provide for enrichment and/or selection for a cell containing nonnative DNA that encodes a protein having a particular feature of interest .
  • the screen preferably can be carried out at increased temperature.
  • screening is done at temperature a few degrees above and a few degrees below the temperature at which the native (i.e., wild-type) alcohol dehydrogenase is inactivated in the particular host cell employed for screening .
  • a protein's activity can be determined by a variety of tests that differ with the various proteins to be tested. A few representative tests that can be employed m the method of the invention are set out m the following Examples. Preferably, however, "activity" means a detectable activity ranging from 10 to 90 units.
  • thermostabilized enzyme might exhibit 10% activity at the same temperature for an increased amount of time, and/or might exhibit an activity at an increased temperature at which the native protein exhibits reduced or no activity.
  • the screening methods also desirably can be done, for instance, in the presence of alcohol, optionally at a lowered pH.
  • Vectors containing mutated versions of the gene of interest optionally can be further mutagenized by repeating steps (b) through (e) above to further stabilize the encoded protein.
  • the present invention accordingly also provides screens that can be employed to select for or against cells having altered ADH activity.
  • the invention provides a method for selecting against growth of Eschericia coli recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Escheri cia coli cells.
  • growth means an increase in cell mass, or some other evidence of cell metabolism such as one of ordinary skill in the art knows how to detect, or is described in the following Examples.
  • An "absence of growth” means growth is not measurable by common procedures (e.g., visual or spectrophotometric observation and the like) or, cell killing. Cell killing can be determined by any well known means, e.g., visual observation, release of cell components, vital staining etc.
  • the E.coli selection method comprises growing said recombinant cells under conditions selected from the group consisting of, wherein ethanol is present in a concentration of about 10%, isopropanol is present in a concentration of about 4%, and propanol is present in a concentration of about 2%, with the proviso that the wild-type cells exhibit reduced or an absence of growth under these conditions.
  • the present invention similarly provides a method for selecting for growth of Thermus flavus recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Thermus flavus cells.
  • This method comprises growing the recombinant cells under conditions selected from the group consisting of wherein ethanol is present at a concentration of aboutl% in a liquid or solid medium at a pH of about 7.0, with the proviso that the wild-type cells exhibit reduced or an absence of growth under these conditions.
  • these methods have been employed to thermostabilize HLADH.
  • the invention provides an isolated and purified thermostabilized HLADH protein comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.
  • the invention also provides genes encoding such protein, e.g., an isolated and purified nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID NO:
  • the invention provides for plasmids encoding for such proteins: e.g., a plasmid comprising one of the aforementioned nucleic acid sequences; and a plasmid selected from the group consisting of pAD7 ; pAD8, pADIO, pAD91, pAD92, pAD93, pAD95, pADlll, pAD113, and pTG450.
  • the invention further preferably provides a method of increasing the thermostability of horse liver alcohol dehydrogenase.
  • This method comprises introducing into a gene which encodes the alcohol dehydrogenase a mutation at a codon which codes for an amino acid residue at a position selected from the group consisting of the amino acid positions, 75, 94, 110, 177, 257, 268, 282, 292, and 297.
  • thermostabilizing the enzyme can be made, for instance, like-for-like (e.g., with acidic amino acids (i.e., aspartic acid, glutamic acid) being substituted for acidic amino acids; basic amino acids (i.e., lysine, arginine, histidine) being substituted for basic amino acids; sulfur containing amino acids (i.e., cysteine) being substituted for sulfur containing amino acids; amides (i.e., asparagine, glutamine) being substituted for amides, aliphatic nonpolar amino acids (i.e., glycine, alanine, valine, leucine, isoleucine) being substituted for aliphatic nonpolar amino acids; and alcoholic, aliphatic, and aromatic amino acids (i.e., serine, threonine, thyrosine, phenylalanine, and tryp
  • EXAMPLE 1 Quantitative assay for ADH in cell extracts. This example describes a method for the quantification of ADH in cell extracts, particularly for the quantitation of HLADH, that can be used according to the invention.
  • EXAMPLE 2 p-Rosanaline/alcohol plate screen in E. coli .
  • This example describes a plate screen for ADH activity that can be employed, for instance, in E. coli .
  • p-Rosaniline indicator plates are prepared according to Conway et al . (Conway et al . , 169, 2591-2597 (1987)) by adding 8 ml of p-rosaniline (2.5 mg/ml in 96% ethanol) and 100 mg of sodium bisulfite to 400 ml batches of precooled (45°C) Luria agar. Most of the dye is immediately converted to the leuco form by reaction with bisulfite to produce a rose-colored medium.
  • Ethanol diffuses into the E. coli cells to produce the acetaldehyde by alcohol dehydrogenase.
  • the leuco dye serves as a sink, reacting with the acetaldehyde to form a Schiff base which is intensely red.
  • the plates can be streaked with a strain or, a strain can be applied in patches to the plate. Colonies will appear a deeper intensity of red dependent upon the level of ADH present in the cell.
  • This example describes a sensitive plate assay of ADH activity which also allows colonies to be tested under different treatment conditions.
  • This assay relies for manipulation of bacterial colonies on the binding of the colonies to a nitrocellulose filter.
  • the assay is carried out by a modified protocol described by Rellos et al . (Rellos et al . , Protein Expression and Purification, _5, 270-277 (1994)) . Namely, a series of temperatures between 65 and 85°C in 5°C increments with incubation times varying from 10 minutes to one hour is analyzed in an attempt to determine the cutoff of the stability of the HLADH protein.
  • the source of the adh gene encoding the HLADH enzyme was plasmid pBPP (Park et al., J. Biol. Chem., 266, 13296-13302 (1991)).
  • E. coli DH5 ⁇ cells containing plasmid pBPP (i.e., HLADH”) or plasmid pCRII (i.e., HLADH ) (InVitrogen; Carlsbad, CA) were grown on rich media plates at cell densities up to about 1,000 colonies per plate and transferred onto a nitrocellulose membrane.
  • the adhered cells were lysed m Buffer 1 (10 mM KMes, pH 6.5 , 0.5 mM C0CI2, 0.1% Triton X-100, 50 ⁇ g/ml lysozyme, 10 ⁇ g/ml DNAse) m a chloroform bath for about one hour, washed once in Buffer 2 (10 mM KMes, 0.5 mM C0CI2, 0.2% BSA), and then washed two more times in Buffer 3 (Buffer 2 without BSA) .
  • Buffer 1 10 mM KMes, pH 6.5 , 0.5 mM C0CI2, 0.1% Triton X-100, 50 ⁇ g/ml lysozyme, 10 ⁇ g/ml DNAse
  • the filters were then incubated at high temperatures m Buffer 4 (10 mM glycme, 0.5 mM C0CI2) and, after washing in Buffer 3, were incubated in the enzyme-detecting solution (30 mM Tris, pH 8.3, 2% ethanol, 1 mM NAD + , 0.1 mg/ml phenazme methosulfate, 1 mg/ml nitroblue tetrazolium) at room temperature for 3-5 minutes .
  • the enzyme-detecting solution (30 mM Tris, pH 8.3, 2% ethanol, 1 mM NAD + , 0.1 mg/ml phenazme methosulfate, 1 mg/ml nitroblue tetrazolium
  • EXAMPLE 4 Shuttle vectors and use of a p-rosanilme assay for verification of the activity of the HLADH gene m Thermus In order to allow expression of the HLADH gene m both Thermus and E. coli , the gene was subcloned into the
  • Thermus shuttle vector pTG100kan tr2 to create plasmid pTG450 depicted Figure 3.
  • the gene is placed upstream of the thermostable kanamycm resistance gene ⁇ kan r2 ) , which is commanded by the lac promoter m E. coli , and the leu promoter m Thermus .
  • An E. coli strain harboring pTG450 has three times more HLADH activity m the presence of IPTG than the strain harboring the original pBPP plasmid.
  • the adh gene integrates into the leuB site m the Thermus chromosome by a double recombination event.
  • Thermus flavus was transformed with both the HLADH plasmid pTG100kan r2 (i.e., creating strain TGF353) and the HLADH + plasmid TG450 (i.e., creating strain TGF650) .
  • TGF650 The presence of the adh gene in TGF650 was confirmed by PCR, and both TGF353 and TGF650 cells were assayed using a variation of the p-rosanilme plate assay described in Example 2.
  • the agar overlay contained the same ingredients described, except TT media (Weber et al . , Bio/Technology, 13, 271-275 (1995); Oshima et al . , International Journal of Systematic Bacteriology, 24, 102-112 (1974) ) was employed instead of Luna broth.
  • TT media Weber et al . , Bio/Technology, 13, 271-275 (1995); Oshima et al . , International Journal of Systematic Bacteriology, 24, 102-112 (1974)
  • a standard p-rosanilme plate can not be used since the indicator dye will spontaneously convert to the Schiff base if incubated overnight the plate as part of this assay.
  • E. coli DH5 cells containing either pTG100kan tr2 (i.e., HLADH”) or pTG450 are examples of E. coli DH5 cells containing either pTG100kan tr2 (i.e., HLADH) or pTG450
  • E . coli cells harboring high activity of HLADH are more sensitive to the presence of the alcohols high concentrations. This probably is due to the accumulation of toxic aldehyde levels m the cells which result from the alcohol dehydrogenase reaction.
  • Three other alcohols were tested (i.e., benzyl alcohol, hexyl alcohol, and hexyl amme) , but did not give clear results because of their poor solubility m the media.
  • results thus confirm that the selection scheme can be employed for the isolation of mutants with altered ADH activity and, m particular, to select against E. coli strains having high levels of ADH.
  • Such a system of negative selection also can be employed to affirmatively identify mutants having high levels of ADH.
  • cells can be replica plated onto a series of plates from a single master plate prior to their transfer to nitrocellulose membranes. One of the plates can be retained, instead of being transferred to nitrocellulose, and matched against the sensitive cells identified in the assay. Cells of interest can then be recovered from the untreated plates .
  • Thermus strains m the presence of the high concentrations of alcohols as a general method for selecting for growth of Thermus strains having high levels of ADH activity.
  • TGF670 HLADH +
  • Thermus rich media e.g., TT media, as described in Oshima et al . , International Journal of Systematic Bacteriology, 24 , 102-112 (1974)
  • TT media as described in Oshima et al . , International Journal of Systematic Bacteriology, 24 , 102-112 (1974)
  • Thermus minimal media Yeh et al . , J. Biol. Chem., 251, 3134-3139 (1976) containing Casaminoacids (TMIN, CAA) .
  • the HLADH+ strain TGF670 demonstrates higher resistance to alcohols than the HLADH " strain TGF353.
  • this selection appears to be dependent on pH, with the selection functioning better at lower pH, especially with ethanol.
  • the selection thus may work by lowering the pH of the media— Thermus prefers higher pH for growth, in the range of pH 7.5-8.5 -- although not enough Thermus biochemistry is known to make this conclusive.
  • EXAMPLE 7 Hydroxylam e mutagenesis of the adh gene. This example describes mutagenesis of the adh gene as a representative alcohol dehydrogenase gene using the mutagen hydroxylamme (HA) .
  • plasmids pBPP and pTG450 both of which contain this gene, were treated with HA using a standard approach. Namely, approximately 8 ⁇ g of plasmid DNA was mixed with 0.5 M NH 2 OH and incubated at 37°C for various lengths of time. For example, aliquots were taken at 1, 2, 3, or 4 hours following treatment, or following overnight exposure to the mutagen. The plasmid DNA was then transformed into
  • Transformants were analyzed by the ADH filter assay described in Example 3, and also using the p-rosanilme assay described m Example 2 to estimate the efficiency of mutagenesis .
  • After overnight treatment only 3 - 4% plasmids treated with HA remained active. Plasmids treated by HA under conditions providing -50% of mactivation of the adh were then transformed into E. coli strain NM554 (obtained from New England Biolabs) to obtain 500 - 700 transformant colonies per plate. These colonies were analyzed by the nitrocellulose filter ADH assay described m Example 3.
  • the filters were incubated for 15 minutes at 70 C in a hybridization oven. Approximately 20,000 transformants were screened using this rapid method. Eighteen candidates were identified which appeared to show increased ADH thermotolerance . The candidates were purified and assayed on the same filter as control strains (i.e., strain XLl containing the LADH * plasmid pBPP, and strain NM554 containing the LADH plasmid pBluesc ⁇ pt) .
  • thermoresistant mutants can be obtained with HA upon further screening.
  • the chances of obtaining mutagenized adh resulting m enzyme thermostabilization might be further increased by excising the mutagenized gene from the vector, and resubclonmg into a wild-type vector (i.e., a vector that has not been treated with HA) , followed by screening.
  • EXAMPLE 8 PCR Mutagenesis of the adh gene This example describes PCR mutagenesis of the adh gene as a representative alcohol dehydrogenase gene. To increase the efficiency of the cloning of mutagenized adh, primers for directional cloning were employed:
  • ADH(Xba ⁇ ) [SEQ ID NO: 22] The adh gene was amplified using these primers and cloned into a pGEM-T vector.
  • Mutagenized adh-containing fragments were digested using Xbal and EcoRI enzymes, and subcloned into pBluescript SK to create a pBlue-ADH library.
  • the resultant pBlue-ADH library i.e., one library for each mutagenesis method performed
  • Transformants were then analyzed: (I) by PCR to determine the efficiency of cloning (% of the plasmids with and without insert) , and ii) by ADH filter assay to determine the efficiency of mutagenesis (% inactive ADH clones) .
  • the results of these analyses are shown m Table 3. Table 3. Mutant candidates identified
  • the transformants were then plated to a density of 500 - 700 cells per plate and assayed on the filters under the same conditions described in the prior example for HA-mutagenesis of the adh gene.
  • thirteen candidates were selected from clones mutagenized by the second method which appeared to possess an HLADH variant that was more stable than the wild-type enzyme.
  • plasmids pAD7 , pAD8 , pADIO, pAD91, pAD92, pAD93, pAD95, pADlll, and pAD113 were chosen for further characterization .
  • thermostable LADH variants confirm that PCR-mediated mutagenesis, particularly as described herein, can be employed to obtain potential thermostable LADH variants.
  • the results further indicate that the method can be employed to obtain other stabilized alcohol dehydrogenases, or other stabilized proteins.
  • HLADH candidates This example describes a characterization for increased thermostability of mutants identified in the prior example.
  • Figure 4 displays the residual activity data for the nine candidate plasmids pAD7 , pAD8 , pADIO, pAD91, pAD92 , pAD93, pAD95, pADlll, and pAD113, wherein the t 0 activity is normalized to 1.00 (100%).
  • all the mutants exhibited increased thermotolerance compared to cells containing plasmid pBPP, which contains the wild-type HLADH gene.
  • plasmids pAD91, pAD92, and pADIO showed the most noticeable alterations in thermostability.
  • This examples describes the sequencing of the mutagenized adh genes.
  • the inserts of plasmids containing the mutagenized adh gene were sequenced using an ABI DNA sequencer, and compared to the sequence of the wild type protein.
  • the translated nucleic acid/amino acid sequence for plasmids having the wild-type or mutant adh genes is given in
  • thermotolerant candidates i.e., those that change the encoded amino acid
  • Table 6 summarizes all the nucleic acid mutations and the respective amino acid changes, if any, introduced by the mutations. Table 6. Mutations identified in thermotolerant candidates
  • pAD7 i.e., nucleic acid sequence at SEQ ID NO : 3 and amino acid sequence at SEQ ID NO:4
  • pAD8 i.e., nucleic acid sequence at SEQ ID NO: 5 and amino acid sequence at SEQ ID NO: 6
  • pADIO i.e., nucleic acid sequence at SEQ ID NO : 7 and amino acid sequence at SEQ ID NO : 8
  • pAD91/pAD92 i.e., nucleic acid sequence at SEQ ID NO : 9 and amino acid sequence at SEQ ID NO: 10
  • pAD93 i.e., nucleic acid sequence at SEQ ID NO: 11 and amino acid sequence at SEQ ID NO:12
  • pAD95 i.e., nucleic acid sequence at SEQ ID NO:13 and amino acid sequence at SEQ ID NO:14
  • pADlll i.e., nucleic acid sequence at SEQ
  • the first numbered am o acid m the wild-type and mutant sequences is serme since, m the sequences studied, the initial methionine (Met) is not present the final protein. However, it is possible that Met is present m the wild-type (or mutant) HLADH sequences that are produced m a different host, e.g., m a eukaryotic host, or when transcribed and translated from a different plasmid construct or chromosome.
  • thermostabilization of HLADH proteins This example describes the means by which the thermostable proteins identified and characterized as in the prior examples can be further thermostabilized. Using the new mutants as a starting point, the process applied here can be reiterated to increase the thermostability of the HLADH enzyme even further. Namely, it is expected that combinations of the identified HLADH mutations or, combinations of these mutations with other HLADH mutations, can further thermostabilize the enzyme.
  • thermoinactivation limits need to be defined as described in Example 3. This is followed by a new round of mutagenesis performed as described in Examples 8, 9, and 10.
  • the identified mutations can be put together in differing combinations by in vi tro site-directed mutagenesis and further molecular biology methods (see, e.g., Sambrook et al . , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY. 1989)) that include DNA shuffling via PCR methods (Stemmer et al . , Proc. Natl. Acad. Sci., 91, 10747-10751 (1994a); Stemmer et al . , Nature, 340, 389-391 (1994b)). As they have done in the past, these methods are all expected to give further increases in the levels of thermostability of the enzyme or, in another similarly screened-for trait.
  • MOLECULE TYPE DNA (genomic)
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC
  • ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC
  • MOLECULE TYPE DNA (genomic)
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC
  • ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370
  • CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110 TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein (ii) SEQUENCE DESCRIPTION SEQ ID NO 16
  • MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO 17 ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480
  • AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC
  • GAA NNN AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA NNN ATT GGT CGG 816 Glu Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa He Gly Arg 260 265 270 CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC NNN CAA GAA GCA TAT GGT 864
  • ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370

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Abstract

The present invention provides a method for the directed evolution of proteins, particularly a method for improving the thermostability of proteins, particularly alcohol dehydrogenases, and especially horse liver alcohol dehydrogenase. The present invention also provides thermostabilized alcohol dehydrogenases produced according to this method.

Description

METHOD FOR THE STABILIZATION OF PROTEINS AND THE THERMOSTABILIZED ALCOHOL DEHYDROGENASES PRODUCED THEREBY
TECHNICAL FIELD OF THE INVENTION The present invention generally relates to a method for the directed evolution of proteins. In particular, the method is directed to stabilization of proteins such as dehydrogenases, and particularly is directed to a method for improving the thermostability of dehydrogenases such as alcohol dehydrogenases. The present invention also relates to thermostabilized alcohol dehydrogenases produced according to this method.
BACKGROUND OF THE INVENTION Biocatalysts are enzymes which can specifically and efficiently expedite chemical reactions such as the synthesis of chemical compounds and biopolymers (Dixon et al . , Enzymes (Academic Press, New York: 1979)) . Biocatalysts are the key players in a number of important industrial synthetic and degradative applications including, but not limited to, the following:
• Synthetic Applications - Biocatalysts currently are employed as feasible alternatives to traditional catalysts, especially for the synthesis of chiral intermediates, or in the reduction of the number of protection/deprotection steps.
• Biodegradation Applications - Biocatalysts currently are employed as enzymatic degradation agents for environmental pollutants such as PCBs, chlorinated hydrocarbons, RDX, halogenated organic compounds, TNT, and other byproducts of industrial production that present significant health risks.
• Diagnostics and Biosensors - Biocatalysts currently are employed as detection agents in diagnostic tests and as biosensors which require enzyme durability.
• Other large-scale industrial applications - Biocatalysts currently are employed as catalysts in the production of fuel supplies through conversion of agricultural feedstocks.
One enzyme that is of considerable utility in current enzymatic processes is the dehydrogenase. In particular, alcohol dehydrogenases are enzymes that command formal, reversible, two-electron chemistry in which alcohols are oxidized to the corresponding ketones. Depending on the precise reaction conditions, ketones can be reduced to the respective alcohols via a stereospecific delivery of a hydride equivalent catalyzed by the enzyme coupled to a bound cofactor such as NADH or NADPH (Lemiere, "Alcohol Dehydrogenase Catalyzed Oxidoreduction Reactions in Organic Chemistry", I_n Enzymes as Catalysts in Organic Synthesis, Schneider et al . , Eds. (1986) p. 17) . This system thus provides a mild, extremely sensitive route to chiral compounds, without contamination from undesired, competing reactions . Such chiral compounds can be used, especially by the pharmaceutical industry, for the preparation of chiral therapeutics, and for effectively generating a wide variety of compounds having the capacity for industrial scale-up (Seebach et al . , Org . Synth . , 63, l-_ (1984); Bradshaw et al . , J. Org. Chem. , 57, 1532(1992); Hummel, Biotechnol . Lett . , 12, 403(1990)). In particular, dehydrogenases show promise for commercial application in the preparation of unusual amino acids and β- hydroxyketones , and in the resolution of racemic alcohols (Benoiton et al . , J. Am. Chem. Soc ■ , 79, 6192 (1957);
Casy et al . , Tetrahedron Lett . , 33 , 817 (1992); Jacovac et al., J. Am. Chem. Soc. , 104, 4659-4665 (1982); Jones et al. Can. J. Chem., 60, 19 (1982)). Of the dehydrogenases, horse liver alcohol dehydrogenase (HLADH) is one of the most commonly used.
For an enzyme biocatalyst such as HLADH to prove useful in a wide-scale, practical, industrial application, it is important that the biocatalyst possess the ability to survive harsh, dynamic, environmental and handling conditions inherent to large-scale commercial processes. These conditions include nonrefπgerated storage, and exposure to organic cosolvents and high reaction temperatures, as well as more idiosyncratic demands imposed by a particular industrial application. To date, one of the greatest challenges associated with biocatalyst implementation is that of overcoming an overall intrinsic instability that results in a requirement for special preparative approaches and handling conditions. Many methods have been used m an attempt to stabilize certain proteins. Rational protein engineering has allowed the redesign of proteins with altered properties such as enhanced stability, shifted pH optima, and different substrate specificities (see, e.g., Bryan et al . , Proteins, 1^, 326-334 (1986); Pantoliano et al . , Biochemistry, 26, 2077-82 (1987); Carter et al . , Science, 237, 394-399 (1987); Wells et al . , "Designing substrate specificity by protein engineering of electrostatic interactions", , 84 , 1219-1223 (1987) ;
Grutter et al . , Nature, 277, 667-669 (1979)).
While potentially an extremely powerful tool, rational protein engineering can be extremely time- consuming and expensive, and currently can be employed only for a very small number of enzymes having well- defined crystal or solution structures. Moreover, since the approach is tailored to a specific enzyme, it typically cannot be generalized to other enzyme species. Other post-production stabilization methods such as immobilization (Macaskie et al . , FEMS Microbiol Rev. , 14_, 351-67 (1994); Shtelzer et al . , Biotechnol . Appl . Biochem. , 15, 227-35 (1992) ; Phadke , Biosystems, 27, 203- 6 (1992)), or use of cross-linked enzymes (Navia et al . , "Crosslmked enzyme crystals as robust biocatalysts" , Proceedings of the Materials Research Society 1993 Symposium, Biomolecular Materials by Design (1993)), suffer some of the same as well as further shortcomings, and similarly, are often too expensive to implement. By contrast, directed evolution potentially can provide a practical approach to tailoring enzymes for a wide range of applications (Shao et al . , "Engineering New Functions and Altering Existing Functions" , Current Opinion in Structural Biology, in press (1996) ) . In support of this, enzymes have been shown to be highly adaptable molecules over evolutionary time scales. Many enzymes catalyzing very different reactions appear to have come about by divergent evolution, acquiring diverse capabilities by the processes of random mutation, recombination, and natural selection.
Thus, there remains a need for an effective means to randomly engineer better enzymes, particularly dehydrogenases, and especially, HLADH. The present invention seeks to overcome some of the aforesaid problems of enzyme design. In particular, it is an object of the present invention to provide a method for the directed evolution of enzymes, particularly dehydrogenases, and especially HLADH. It further is an object of the present invention to provide a method for stabilizing, e.g. improving the thermostability of enzymes such as dehydrogenases . Such a method of stabilizing dehydrogenases (particularly HLADH) would present a major advancement in the field since it would extend the shelf life, longevity, and active temperature range of these enzymes. These and other objects and advantages of the present invention, as well as further inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
Briefly, the present invention provides, inter alia , a method for the stabilization of a protein (particularly for the stabilization of an alcohol dehydrogenase such as horse liver alcohol dehydrogenase (HLADH) , general enrichment/selection means that can be employed in Escherichia and Thermus to select for cells having altered levels of alcohol dehydrogenase activity as compared to a wild-type cell, thermostabilized HLADH proteins and nucleic acid sequences encoding same, as well as plasmids and hosts cells comprising the nucleic acid sequences .
BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagram that generally depicts the approach of the present invention for the accelerated evolution of enzymes. A pool of mutants of the particular gene is obtained by means such as spontaneous, directed, chemical, or PCR-mediated mutagenesis. The mutants of interest (i.e., having the particular stabilized feature) are identified by means of a screen or selection (A) , and optionally, compatible mutations can be combined (e.g., by gene splicing, in vi tro recombination, and the like) to enhance the stability even further (B) .
Figure 2 is a digitized image of results of a filter assay for alcohol dehydrogenase activity which demonstrates that wild-type HLADH is rapidly inactivated at 75 °C: no heat treatment (A) ; 5 minutes of heat treatment at 75 °C (B) ; 10 minutes of heat treatment at 75 °C (C) ; 15 minutes of heat treatment at 75 °C (D) ; 20 minutes of heat treatment at 75 °C (E) ; and 50 minutes of heat treatment at 75 °C (F) .
Figure 3 is a partial restriction map of the plasmid pTG450 which contains the adh gene from plasmid pBPP cloned into a pTG100kantr2 Thermus shuttle vector.
Figure 4 is a bar chart that depicts the increased thermostability of HLADH mutants produced according to the invention at 70°C. Cells containing pGEM-T (i.e., having no HLADH gene) did not show any HLADH activity. Figure 5 is the sequence of adh gene [SEQ ID NO:l] that encodes the HLADH protein [SEQ ID NO: 2] , with the location of certain mutations produced according to the invention identified as the boxed regions.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides, among other things, a method for stabilizing a certain feature of a protein (e.g., stability at a certain temperature, stability in the presence of certain reagents, etc.) . In particular, the method of the invention provides a method for thermostabilizing a protein. Namely, the invention preferably provides a method of obtaining nonnative protein having a thermostability that is increased over that of the native version of said protein, as further described herein. According to the invention, a "native" protein is the protein as it generally is found in nature. By contrast, a "nonnative" protein differs from the native protein in that it has been modified by human intervention, i.e., at either the level of the protein or its encoding DNA (e.g., by recombinant means to directly alter the genome; by unique selection and forced mutation; by random mutagenesis) . Moreover, a "protein" desirably can be either an entire protein, or a portion of a protein (e.g., as where a chimeric nonnative protein results from either transcriptional or translational gene fusion) . Similarly, a "nonnative protein" in some applications (e.g., applications for further study) may be a peptide (i.e., an incomplete protein) , as where the peptide is chemically synthesized or, where a gene's coding sequence is transcribed or translated in vi tro or, is produced by chemical processing of a complete protein.
A preferred protein for stabilization, particularly thermostabilization according to the invention is a dehydrogenase, particularly an alcohol dehydrogenase, and especially horse liver alcohol dehydrogenase (e.g., as obtained from plasmid pBPP, and/or as set forth in SEQ ID NO: 2) . Notably, with respect to SEQ ID NO: 2, this protein does not initiate with methionine (Met) . However, other varients of horse liver alcohol dehydrogenase produced by in vi tro synthetic reactions, by means of chemical synthesis or, in other hosts (e.g., an eukaryotic host or other prokaryotic host cell) may possess a methionine residue in the first position of the protein. The numbering of residues in such proteins of course, would differ somewhat from that of SEQ ID NO:2. Namely, the second position of the aforementioned protein would be equivalent to the first position of the protein of SEQ ID NO : 2. Of course, the ordinarily skilled artisan would know how to compare equivalent regions of proteins . Desirably, other proteins (particularly proteins having capacity for industrial implementation) can be stabilized (e.g., thermostabilized) according to the invention. For instance, an alcohol dehydrogenase protein can be employed from another species. It is anticipated that this approach can be employed with alcohol dehydrogenases from other species based on the similarities between certain of the various alcohol dehydrogenases. Also, a protein according to the invention optionally can be another type of dehydrogenase, e.g., another type of NAD+ (P) -linked dehydrogenase including, but not limited to, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase (NADP+) , hydroxylacyl CoA dehydrogenase, glyceraldehyde 3 -phosphate dehydrogenase, and glucose 6- phosphate dehydrogenase (NADP+) .
In a preferred embodiment, the method can be employed to thermostabilize a horse liver alcohol dehydrogenase. This method generally is depicted in Figure 1. Preferably the method comprises: (a) obtaining in a vector a gene that encodes the native protein; (b) mutating the vector at more than one position in the gene to produce a vector library of cells comprising mutated versions of the gene;
(c) introducing the vector library en masse into cells of a strain in which the majority of the mutated versions of the gene are transcribed and translated to produce a cell library;
(d) screening the cell library to identify a cell comprising a mutated version of the gene that encodes a nonnative protein having a thermostability that is increased over that of the wild-type verson of the protein; and
(e) purifying the cell from the cell library. According to the invention, "gene that encodes said protein" can comprise a recombinant or nonrecombinant sequence, i.e., a sequence that is present as found in nature (i.e., encodes a native amino acid sequence) or, has been modified, for instance by the introduction of mutations (e.g., point mutations, insertions, deletions, or rearrangements) to comprise a nonnative amino acid sequence or, can be a mixture of native and nonnative amino acid sequences. Similarly, a recombinant gene may conjoin coding sequences (either in entirety or in part) with regulatory sequences (e.g., transcription initiation, transcription termination, translational start or stop sites, protein secretion sequences, and the like) which are not typically conjoined in nature. This can allow the production of a protein in a host in which it normally is not produced (e.g., production of a eukaryotic protein in a prokaryotic cell) . Preferably, however, the recombinant gene (which can derive, in entirety or part, from any prokaryotic, eukaryotic, bacteriophage , or viral source) is capable of being transcribed and translated in a prokaryotic cell, particularly, a cell comprising a member of the genuses Escherichi or Thermus . Thus, preferably a host cell in the context of the present invention (i.e., which can be employed in a method of stabilizing proteins) is a member of the kingdom Bacteria, Archaea, or Eukarya . In particular, preferably a cell employed in the method of stabilizing (particularly thermostabilizing) proteins according to the invention is a thermophile or hyperthermophile . In particular, preferably a cell is a member of the genus Thermus , and desirably is of the species Thermus flavus, Thermus aqua ticus, Thermus thermophilus, or Thermus sp . Optimally a cell is either an Escherichia coli cell or a Thermus aquaticus cell.
The vector in which the gene of interest is subcloned can be any vector appropriate for delivery of a gene to a cell. For instance, the vector can be a plasmid, bacteriophage, virus, phagemid, cointegrate of one or more vector species, etc. Optimally, however, a vector is one that can be employed for gene expression in a prokaryotic cell such as a Thermus or Eshcerichia cell. It also is preferable that a vector have an ability to shuttle between different cells, e.g., between a Thermus and an Eschericia cell. One such vector that can be employed in the context of the invention is the vector pTG450. The preferred method of the invention calls for mutating a vector containing the gene encoding the protein to be stabilized. Any method of mutagenesis such as is known to those skilled in the art and particularly as is described in the following Examples, can be employed in the method of the invention for generating a mutated gene. Desirably a PCR-based (error prone) approach, especially as set out as follows, is employed for mutagenesis. However, other mutagens (e.g., chemical mutagens such as hydroxylamine) , also can be employed. In the preferred method of mutagenesis employed in the invention, desirably the vector is mutated at more than one position in the gene of interest. This can be assessed by means known in the art and as described in the Examples. Such mutagenesis in more than one position in the gene will result in a "vector library" comprising mutated versions of a gene, particularly of a horse liver alcohol dehydrogenase gene, which are present in the library mixture.
The vector library can be introduced en masse into cells (e.g., by transformation) . Since the vectors and the cells employed for these methods are selected to be compatible, and the gene is engineered (e.g., as described below) to contain or to be flanked by any sequences necessary for its expression, it is expected that such introduction will result in the transcription and ensuing translation of the introduced gene. Moreover, such en masse introduction will result in the generation of a cell library comprising a mixture of cells transformed with plasmids having differing mutated genes. In some instances, it may be desirable to reisolate the vectors from the cell library (e.g., by a plasmid isolation or other vector isolation protocol) , excise out the mutated gene, and subclone the mutated gene into another vector (e.g., a vector that has not been mutagenized) .
Following the generation of the cell library, the cells preferably are screened under conditions that allow identification of a cell comprising a mutated version of the gene of interest that encodes a nonnative protein having a protein that is stabilized (e.g., thermostabilized) over that of the wild-type (i.e., native) versions of the protein. A variety of selection means can be employed in accordance with the method of the present invention and, in particular, the selection means identified in the Examples which follow can be employed. Of course, one of ordinary skill in the art could modify these methods such that they are adapted for a particular host cell and/or a particular protein of interest. Desirably, however, screening conditions are employed that provide for enrichment and/or selection for a cell containing nonnative DNA that encodes a protein having a particular feature of interest . In particular, when the protein being stabilized according to the invention is an alcohol dehydrogenase, and particularly HLADH, the screen preferably can be carried out at increased temperature. For instance, desirably, screening is done at temperature a few degrees above and a few degrees below the temperature at which the native (i.e., wild-type) alcohol dehydrogenase is inactivated in the particular host cell employed for screening .
According to this invention, "increasing the thermostability" of a nonnative protein means: (a) increasing the length of time at which a nonnative protein exhibits activity as compared to the wild-type protein; (b) increasing the temperature at which a nonnative protein exhibits activity as compared to a wild-type protein; or (c) increasing the length of time and temperature at which a nonnative protein exhibits activity as compared to a wild-type protein. A protein's activity can be determined by a variety of tests that differ with the various proteins to be tested. A few representative tests that can be employed m the method of the invention are set out m the following Examples. Preferably, however, "activity" means a detectable activity ranging from 10 to 90 units. For instance, whereas a wild-type protein might exhibit 10% activity at a defined temperature for a set amount of time, a thermostabilized enzyme might exhibit 10% activity at the same temperature for an increased amount of time, and/or might exhibit an activity at an increased temperature at which the native protein exhibits reduced or no activity. The screening methods also desirably can be done, for instance, in the presence of alcohol, optionally at a lowered pH.
Following screening of cells to identify those having the desired trait (s) imparted by the mutated gene, optionally, cells exhibiting the trait can be further isolated. Vectors containing mutated versions of the gene of interest optionally can be further mutagenized by repeating steps (b) through (e) above to further stabilize the encoded protein.
The present invention accordingly also provides screens that can be employed to select for or against cells having altered ADH activity. For instance, the invention provides a method for selecting against growth of Eschericia coli recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Escheri cia coli cells. According to this invention, "growth" means an increase in cell mass, or some other evidence of cell metabolism such as one of ordinary skill in the art knows how to detect, or is described in the following Examples. An "absence of growth" means growth is not measurable by common procedures (e.g., visual or spectrophotometric observation and the like) or, cell killing. Cell killing can be determined by any well known means, e.g., visual observation, release of cell components, vital staining etc.
Thus the E.coli selection method comprises growing said recombinant cells under conditions selected from the group consisting of, wherein ethanol is present in a concentration of about 10%, isopropanol is present in a concentration of about 4%, and propanol is present in a concentration of about 2%, with the proviso that the wild-type cells exhibit reduced or an absence of growth under these conditions.
The present invention similarly provides a method for selecting for growth of Thermus flavus recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Thermus flavus cells. This method comprises growing the recombinant cells under conditions selected from the group consisting of wherein ethanol is present at a concentration of aboutl% in a liquid or solid medium at a pH of about 7.0, with the proviso that the wild-type cells exhibit reduced or an absence of growth under these conditions. As mentioned previously, these methods have been employed to thermostabilize HLADH. In particular, the invention provides an isolated and purified thermostabilized HLADH protein comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20. The invention also provides genes encoding such protein, e.g., an isolated and purified nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.
Moreover, the invention provides for plasmids encoding for such proteins: e.g., a plasmid comprising one of the aforementioned nucleic acid sequences; and a plasmid selected from the group consisting of pAD7 ; pAD8, pADIO, pAD91, pAD92, pAD93, pAD95, pADlll, pAD113, and pTG450.
The invention further preferably provides a method of increasing the thermostability of horse liver alcohol dehydrogenase. This method comprises introducing into a gene which encodes the alcohol dehydrogenase a mutation at a codon which codes for an amino acid residue at a position selected from the group consisting of the amino acid positions, 75, 94, 110, 177, 257, 268, 282, 292, and 297. Examination of the three-dimensional structure of the HLADH protein will elucidate the manner in which further amino acid substitutions thermostabilizing the enzyme can be made, for instance, like-for-like (e.g., with acidic amino acids (i.e., aspartic acid, glutamic acid) being substituted for acidic amino acids; basic amino acids (i.e., lysine, arginine, histidine) being substituted for basic amino acids; sulfur containing amino acids (i.e., cysteine) being substituted for sulfur containing amino acids; amides (i.e., asparagine, glutamine) being substituted for amides, aliphatic nonpolar amino acids (i.e., glycine, alanine, valine, leucine, isoleucine) being substituted for aliphatic nonpolar amino acids; and alcoholic, aliphatic, and aromatic amino acids (i.e., serine, threonine, thyrosine, phenylalanine, and tryptophan) being substituted for alcoholic, aliphatic, and aromatic amino acids .
Additional uses and benefits of the invention will be apparent to one of ordinary skill in the art.
EXAMPLES
The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLE 1 : Quantitative assay for ADH in cell extracts. This example describes a method for the quantification of ADH in cell extracts, particularly for the quantitation of HLADH, that can be used according to the invention.
For this assay, overnight cultures of cells to be assayed are grown in rich media. The cells are washed, resuspended in 600 μl of assay buffer (83 mM KH2PO4 [pH 7.3], 40 mM KC1 , 0.25 mM EDTA), and sonicated. The assay mixture contains 500 μl of cell extract, 100 μl EtOH, 20 μl 100 mM NAD, 830 μl buffer and is carried out at room temperature. The reaction is run for 3 minutes and absorbence at 340 nM is measured. Using this approach it is possible to identify a high IPTG inducible activity in the strains with the HLADH coding sequence under the control of the lacZ promoter. This method thus produces a reliable quantitative determination of HLADH activity present in the cell .
EXAMPLE 2 : p-Rosanaline/alcohol plate screen in E. coli . This example describes a plate screen for ADH activity that can be employed, for instance, in E. coli . p-Rosaniline indicator plates are prepared according to Conway et al . (Conway et al . , 169, 2591-2597 (1987)) by adding 8 ml of p-rosaniline (2.5 mg/ml in 96% ethanol) and 100 mg of sodium bisulfite to 400 ml batches of precooled (45°C) Luria agar. Most of the dye is immediately converted to the leuco form by reaction with bisulfite to produce a rose-colored medium. Ethanol diffuses into the E. coli cells to produce the acetaldehyde by alcohol dehydrogenase. The leuco dye serves as a sink, reacting with the acetaldehyde to form a Schiff base which is intensely red. Thus, the plates can be streaked with a strain or, a strain can be applied in patches to the plate. Colonies will appear a deeper intensity of red dependent upon the level of ADH present in the cell. In particular, by plating appropriate controls on each plate, it is relatively easy to visually discern a strain which has a high level of dehydrogenase (deep red staining) , an intermediate level of dehydrogenase (more moderate red staining) , and no activity (little or no red staining) . This method thus provides a plate screen that can be employed in the method of the invention. EXAMPLE 3 : Filter screen for HLADH activity.
This example describes a sensitive plate assay of ADH activity which also allows colonies to be tested under different treatment conditions. This assay relies for manipulation of bacterial colonies on the binding of the colonies to a nitrocellulose filter. The assay is carried out by a modified protocol described by Rellos et al . (Rellos et al . , Protein Expression and Purification, _5, 270-277 (1994)) . Namely, a series of temperatures between 65 and 85°C in 5°C increments with incubation times varying from 10 minutes to one hour is analyzed in an attempt to determine the cutoff of the stability of the HLADH protein. For these experiments, the source of the adh gene encoding the HLADH enzyme was plasmid pBPP (Park et al., J. Biol. Chem., 266, 13296-13302 (1991)).
E. coli DH5α cells containing plasmid pBPP (i.e., HLADH") or plasmid pCRII (i.e., HLADH ) (InVitrogen; Carlsbad, CA) were grown on rich media plates at cell densities up to about 1,000 colonies per plate and transferred onto a nitrocellulose membrane. The adhered cells were lysed m Buffer 1 (10 mM KMes, pH 6.5 , 0.5 mM C0CI2, 0.1% Triton X-100, 50 μg/ml lysozyme, 10 μg/ml DNAse) m a chloroform bath for about one hour, washed once in Buffer 2 (10 mM KMes, 0.5 mM C0CI2, 0.2% BSA), and then washed two more times in Buffer 3 (Buffer 2 without BSA) . The filters were then incubated at high temperatures m Buffer 4 (10 mM glycme, 0.5 mM C0CI2) and, after washing in Buffer 3, were incubated in the enzyme-detecting solution (30 mM Tris, pH 8.3, 2% ethanol, 1 mM NAD+, 0.1 mg/ml phenazme methosulfate, 1 mg/ml nitroblue tetrazolium) at room temperature for 3-5 minutes .
Results of these experiments are depicted m Figure 2. As can be seen in this figure, the experiments confirm that a 15-20 minute treatment of the filters at
75°C resulted m roughly 90% inactivation of the HLADH protein as estimated by the color changes. This information on the activity of the native protein can be used as a baseline for the identification and isolation of mutagenized candidates having altered ADH activity according to the invention.
EXAMPLE 4 : Shuttle vectors and use of a p-rosanilme assay for verification of the activity of the HLADH gene m Thermus In order to allow expression of the HLADH gene m both Thermus and E. coli , the gene was subcloned into the
Thermus shuttle vector, pTG100kantr2 to create plasmid pTG450 depicted Figure 3. In this construct, the gene is placed upstream of the thermostable kanamycm resistance gene {kan r2 ) , which is commanded by the lac promoter m E. coli , and the leu promoter m Thermus .
An E. coli strain harboring pTG450 has three times more HLADH activity m the presence of IPTG than the strain harboring the original pBPP plasmid. When transformed into Thermus , the adh gene integrates into the leuB site m the Thermus chromosome by a double recombination event. For these experiments, Thermus flavus was transformed with both the HLADH plasmid pTG100kan r2 (i.e., creating strain TGF353) and the HLADH+ plasmid TG450 (i.e., creating strain TGF650) .
The presence of the adh gene in TGF650 was confirmed by PCR, and both TGF353 and TGF650 cells were assayed using a variation of the p-rosanilme plate assay described in Example 2. Namely, the agar overlay contained the same ingredients described, except TT media (Weber et al . , Bio/Technology, 13, 271-275 (1995); Oshima et al . , International Journal of Systematic Bacteriology, 24, 102-112 (1974) ) was employed instead of Luna broth. A standard p-rosanilme plate can not be used since the indicator dye will spontaneously convert to the Schiff base if incubated overnight the plate as part of this assay. Using this approach, HLADH activity was observed in the pTG450 Thermus transformants at a level well above background levels observed for the pTG100kantr2 Thermus transformants . The activity was observed up to 70 °C. These results thus confirm that a p-rosaniline plate assay similarly can be employed in the context of the present invention for screening in Thermus for mutants having altered ADH activity.
EXAMPLE 5: Development of a Method of HLADH
Selection/Enrichment in E. coli This example describes a method of negative selection for growth of E. coli strains harboring the adh gene . For these experiments, E. coli DH5 cells containing either pTG100kantr2 (i.e., HLADH") or pTG450
(i.e., HLADH+) were grown on LB plates with different alcohols in concentrations ranging from 2% to 12%. The results of one such experiment are displayed in Table 1.
Table 1. Effect of varying concentrations of alcohol in Escherichia coli
Figure imgf000021_0001
n x m
[TJ DH5α 2 4 8 10 12 2 4 8 10 12 2 4 6 8 12 2 4 8 12
33
C r- PTG100kantr2 ++ ++ ++ ++ ++ ++ ++ ++ - - + ++ +- ++ m t σ>
PTG450 ++ ++ + + +- ++ ++ +- - - + +- -. _ _ +_
Symbols in order of decreasing growth: ++, +, +-, -
As can be seen from Table 1, E . coli cells harboring high activity of HLADH (i.e., transformed with the HLADH* plasmid pTG450) are more sensitive to the presence of the alcohols high concentrations. This probably is due to the accumulation of toxic aldehyde levels m the cells which result from the alcohol dehydrogenase reaction. Three other alcohols were tested (i.e., benzyl alcohol, hexyl alcohol, and hexyl amme) , but did not give clear results because of their poor solubility m the media.
The experiment was repeated several times and the alcohol levels were refined to determine a range resulting a clear selection. Three of the alcohols, i.e., ethanol at a concentration of 10%, isopropanol at a concentration of 4%, and propanol at a concentration of 2%, resulted in clean, negative selection for growth of E. coli harboring the adh gene.
These results thus confirm that the selection scheme can be employed for the isolation of mutants with altered ADH activity and, m particular, to select against E. coli strains having high levels of ADH. Such a system of negative selection also can be employed to affirmatively identify mutants having high levels of ADH. For instance, cells can be replica plated onto a series of plates from a single master plate prior to their transfer to nitrocellulose membranes. One of the plates can be retained, instead of being transferred to nitrocellulose, and matched against the sensitive cells identified in the assay. Cells of interest can then be recovered from the untreated plates .
EXAMPLE 6 : Development of a Method of HLADH
Selection/Enrichment Thermus This example describes the growth of Thermus strains m the presence of the high concentrations of alcohols as a general method for selecting for growth of Thermus strains having high levels of ADH activity.
A series of experiments was conducted to develop a selection using alcohol levels in Thermus . In these experiments, Thermus flavus strains TGF353 (HLADH") and
TGF670 (HLADH+) were employed. Each strain was grown for two days on Thermus rich media (e.g., TT media, as described in Oshima et al . , International Journal of Systematic Bacteriology, 24 , 102-112 (1974) ) present in plates or, was grown overnight in 4 ml of liquid TT medium, in order to ensure the cells were at the same physiological stage prior to testing. The test itself was performed on TT media and Thermus minimal media (Yeh et al . , J. Biol. Chem., 251, 3134-3139 (1976) containing Casaminoacids (TMIN, CAA) . Over a series of many experiments, the strains were grown on agar plates or in liquid medium containing various concentrations of ethanol (i.e., 0.5, 1, 2, 4, 6, or 8%), various concentrations of methanol (i.e., 2, 4, 6, or 8%), various concentrations of isopropanol (i.e., 0.5, 1, 2, 4, or 6%), various concentrations of propanol (i.e., 1, 2, 4, or 6%), or various concentrations of propanediol (i.e. 0.5 or 1%) . Such experiments further were done at different pHs, i.e., at pH 7.0, 7.5 and 8.0, for the various alcohols at different concentrations. The results of one of these experiments is set out in Table 2.
Table 2. Optical density (OD600) in various media
Figure imgf000024_0001
c J t ι- m ro
Figure imgf000024_0002
As can be seen from this experiment, the HLADH+ strain TGF670 demonstrates higher resistance to alcohols than the HLADH" strain TGF353. Moreover, this selection appears to be dependent on pH, with the selection functioning better at lower pH, especially with ethanol. The selection thus may work by lowering the pH of the media— Thermus prefers higher pH for growth, in the range of pH 7.5-8.5 -- although not enough Thermus biochemistry is known to make this conclusive.
A similar effect can also be achieved on plates. However, the primary effect of the screen in Thermus is to retard growth of cells without the adh gene, not to completely eliminate it. This also is the case with the liquid media, indicating that a completely clean selection m Thermus without background is difficult to achieve. Nevertheless, this selection means provides a powerful enrichment, especially in liquid, by selecting for faster growing cells under the conditions defined. The results thus confirm that the enrichment/selection means outlined above can be employed with Thermus .
EXAMPLE 7 : Hydroxylam e mutagenesis of the adh gene. This example describes mutagenesis of the adh gene as a representative alcohol dehydrogenase gene using the mutagen hydroxylamme (HA) .
For HA mutagenesis of the adh gene, plasmids pBPP and pTG450, both of which contain this gene, were treated with HA using a standard approach. Namely, approximately 8 μg of plasmid DNA was mixed with 0.5 M NH2OH and incubated at 37°C for various lengths of time. For example, aliquots were taken at 1, 2, 3, or 4 hours following treatment, or following overnight exposure to the mutagen. The plasmid DNA was then transformed into
E. coli strain DH5α and plated onto LBA 100 plates (i.e. LB plates containing 100 μg/ml ampicillin) . Transformants were analyzed by the ADH filter assay described in Example 3, and also using the p-rosanilme assay described m Example 2 to estimate the efficiency of mutagenesis . After overnight treatment, only 3 - 4% plasmids treated with HA remained active. Plasmids treated by HA under conditions providing -50% of mactivation of the adh were then transformed into E. coli strain NM554 (obtained from New England Biolabs) to obtain 500 - 700 transformant colonies per plate. These colonies were analyzed by the nitrocellulose filter ADH assay described m Example 3. For heat mactivation of ADH, the filters were incubated for 15 minutes at 70 C in a hybridization oven. Approximately 20,000 transformants were screened using this rapid method. Eighteen candidates were identified which appeared to show increased ADH thermotolerance . The candidates were purified and assayed on the same filter as control strains (i.e., strain XLl containing the LADH* plasmid pBPP, and strain NM554 containing the LADH plasmid pBluescπpt) .
Based on results of the filter screening, none of the identified candidates appeared to have the temperature-resistant phenotype suggested by the results of the ADH filter assay. It is possible, however, that thermoresistant mutants can be obtained with HA upon further screening. Moreover, the chances of obtaining mutagenized adh resulting m enzyme thermostabilization might be further increased by excising the mutagenized gene from the vector, and resubclonmg into a wild-type vector (i.e., a vector that has not been treated with HA) , followed by screening.
EXAMPLE 8 : PCR Mutagenesis of the adh gene This example describes PCR mutagenesis of the adh gene as a representative alcohol dehydrogenase gene. To increase the efficiency of the cloning of mutagenized adh, primers for directional cloning were employed:
CCC CGA ATT CTC AAA ACG TCA GGA TGG TAC G ADH(EcoRI) [SEQ ID NO: 21]
CCC CTC TAG AAT AAA TGA GCA CAG CAG GAA AAG TAA TAA AAT GC
ADH(XbaΙ) [SEQ ID NO: 22] The adh gene was amplified using these primers and cloned into a pGEM-T vector.
For PCR mutagenesis two protocols were used, one according to Spee et al . (Spee et al . , Nucl . Acids Res . , 21, 777-778 (1993)), and another according to Rellos et al . , (Rellos et al . , supra) m which the limiting dNTP concentration was double that of the first procedure and dITP was not employed. The pGEM-T plasmid containing the adh gene was then used as a template for PCR mutagenesis of adh using standard T7 and SP6 primers to perform the error-prone PCR reaction under these conditions. Mutagenized adh-containing fragments were digested using Xbal and EcoRI enzymes, and subcloned into pBluescript SK to create a pBlue-ADH library. The resultant pBlue-ADH library (i.e., one library for each mutagenesis method performed) was transformed en masse into E. coli strain NM554 to allow the adh gene to be transcribed from the lac promoter. Transformants were then analyzed: (I) by PCR to determine the efficiency of cloning (% of the plasmids with and without insert) , and ii) by ADH filter assay to determine the efficiency of mutagenesis (% inactive ADH clones) . The results of these analyses are shown m Table 3. Table 3. Mutant candidates identified
Method of Percentage of the Percentage of the mutagenesis* plasmids with the ADH+ clones insert
Method No. 1 60% 64%
Method No. 2 90% 36%
No mutagenesis 80% 75% (wild-type adh)
* Method No .1 was done according to Spee et al . , supra , (i.e. with 14 μM of limiting dNTP and 200 μM dITP) and
Method No. 2 was done according to Rellos et al . , supra (i.e. without dITP and with 25 μM of the limiting dNTP)
As can be seen from these results, both the cloning and mutagenesis efficiency was better using the second method.
The transformants were then plated to a density of 500 - 700 cells per plate and assayed on the filters under the same conditions described in the prior example for HA-mutagenesis of the adh gene. Approximately 5,000 clones containing adh mutagenized by the first method, and the same number of clones mutagenized by the second method, were tested. No thermostable candidates from the first method were identified. By contrast, thirteen candidates were selected from clones mutagenized by the second method which appeared to possess an HLADH variant that was more stable than the wild-type enzyme. Upon restreaking and retesting these colonies by the filter assay method, nine of the thirteen candidates (i.e., plasmids pAD7 , pAD8 , pADIO, pAD91, pAD92, pAD93, pAD95, pADlll, and pAD113) were chosen for further characterization .
These results confirm that PCR-mediated mutagenesis, particularly as described herein, can be employed to obtain potential thermostable LADH variants. The results further indicate that the method can be employed to obtain other stabilized alcohol dehydrogenases, or other stabilized proteins.
EXAMPLE 9 : Characterization of thermotolerant
HLADH candidates . This example describes a characterization for increased thermostability of mutants identified in the prior example.
These experiments were done by calculating the residual HLADH activity at 70°C for a series of incubation periods. Residual activity is calculated as activity after incubation at a particular temperature divided by activity before incubation. Cultures of the mutant candidates as well as control cells harboring the wild-type HLADH+ control plasmid pBPP and HLADH negative control plasmid pGEM-T were grown m appropriate media, and cell extracts were made by somcation. The extracts were then incubated at 70°C, taking an initial sample
(t ) , and sampling at about 30, 60, and 120 minutes. The samples were stored on ice, and the HLADH activity was determined spectrophotometrically as described in Example 1. The data was plotted as a percentage of activity compared to the t0 activity (residual activity) in order to compare the individual samples to each other and ad ust for variations in expression levels or growth variations .
Figure 4 displays the residual activity data for the nine candidate plasmids pAD7 , pAD8 , pADIO, pAD91, pAD92 , pAD93, pAD95, pADlll, and pAD113, wherein the t0 activity is normalized to 1.00 (100%). As can be seen from Figure 4, all the mutants exhibited increased thermotolerance compared to cells containing plasmid pBPP, which contains the wild-type HLADH gene. In particular, plasmids pAD91, pAD92, and pADIO showed the most noticeable alterations in thermostability. Cells containing pGEM-T (i.e., not having an HLADH gene) did not show any HLADH activity. These results thus confirm that the method of the invention can be employed to obtain thermostable alcohol dehydrase, particularly HLADH, mutants.
Table 4 below provides data illustrating comparative data for HALDH activities in the original wild-type ( "WT" ) clone and mutants. All clones were grown in 50 ml of LB medium with 100 μg/ml Amp (12.5 μg/ml Tet for WT clone) overnight, concentrated in 1 ml of the assay buffer (83 mM KH2P04, 40 mM KCl, 0.25 mM EDTA), sonicated and assayed with ethanol as a substrate and NAD cofactor, with results shown as U = mol/mg protein x 1000 / percent residual activity.
Table 4. HALDH Activity after Heat Treatment
Heat Treatment time
Figure imgf000030_0001
Table 5 below provides data illustrating comparative data for HALDH activities of the original wild-type ( "WT" ) clone and mutants and substrate specificity. All clones were grown in 1 L of LB medium with 100 μg/ml Amp (12.5 μg/ml Tet for WT clone) overnight, concentrated in 50 ml of the assay buffer (83 mM KH2P04, 40 mM KCl, 0.25 mM EDTA) , sonicated, incubated at 55°C for 5 min to denature the E. coli protiens and lyophilized. The assays were performed at room temperature with the listed substrate and NAD cofactor, with results shown as U = mol/mg protein x 1000. Table 5 HLADH Substrate Speci f icity
Strain Ethanol Isopropanol Butanol Benzyl Alcol
Figure imgf000031_0001
EXAMPLE 10 Sequence Analysis of HLADH Thermotolerant Candidates
This examples describes the sequencing of the mutagenized adh genes.
The inserts of plasmids containing the mutagenized adh gene were sequenced using an ABI DNA sequencer, and compared to the sequence of the wild type protein. The translated nucleic acid/amino acid sequence for plasmids having the wild-type or mutant adh genes is given in
Figure 5, with the positions of the non-silent mutations (i.e., those that change the encoded amino acid) indicated by the boxes. Table 6 summarizes all the nucleic acid mutations and the respective amino acid changes, if any, introduced by the mutations. Table 6. Mutations identified in thermotolerant candidates
Mutant Base Amino Original Mutant Amino plasmid pair acid codon codon acid position position1 change2 pAD7 774 257 ATG ATA Met257Ile
878 292 GTG GCG Val292Ala pAD8 285 94 ACT ACC no aa change
806 268 GTC GCC Val268Ala pADIO 227 75 AGC AAC Ser75Asn pAD91/92 284 94 ACT ATT Thr94Ile pAD93 847 282 TGT AGT Cys282Ser
893 297 GAT GGT Asp297Gly pAD95 774 257 ATG ATA Met257Ile
878 292 GTG GCG Val292Ala pADlll 532 177 TCT ACT Serl77Thr pAD113 129 42 GCC GCT no aa change
159 52 GTG GTA no aa change
331 110 TTC CTC PhellOLeu
Also, the individual sequences of the mutant adh sequences are set forth in the Sequence Listing for pAD7 (i.e., nucleic acid sequence at SEQ ID NO : 3 and amino acid sequence at SEQ ID NO:4), pAD8 (i.e., nucleic acid sequence at SEQ ID NO: 5 and amino acid sequence at SEQ ID NO: 6), pADIO (i.e., nucleic acid sequence at SEQ ID NO : 7 and amino acid sequence at SEQ ID NO : 8 ) , pAD91/pAD92 (i.e., nucleic acid sequence at SEQ ID NO : 9 and amino acid sequence at SEQ ID NO: 10), pAD93 (i.e., nucleic acid sequence at SEQ ID NO: 11 and amino acid sequence at SEQ ID NO:12), pAD95 (i.e., nucleic acid sequence at SEQ ID NO:13 and amino acid sequence at SEQ ID NO:14), pADlll (i.e., nucleic acid sequence at SEQ ID NO: 15 and ammo acid sequence at SEQ ID NO: 16), and pAD113(ι.e., nucleic acid sequence at SEQ ID NO: 17 and ammo acid sequence at SEQ ID NO: 18) . The first numbered am o acid m the wild-type and mutant sequences is serme since, m the sequences studied, the initial methionine (Met) is not present the final protein. However, it is possible that Met is present m the wild-type (or mutant) HLADH sequences that are produced m a different host, e.g., m a eukaryotic host, or when transcribed and translated from a different plasmid construct or chromosome.
As can be seen from this data, the sequences of pAD91 and pAD92 are identical, which indicates the clones from which the DNA was isolated likely are siblings. Mutants containing plasmids pAD91, PAD92 , pAD93, and pAD95 were identified from the same filter and mutants containing plasmids pADlll and pAD113 were identified from the same filter assay. Also, both pAD8 and pAD91/92, the coding sequence specifying ammo acid 94 is mutated. Whereas this results in no change m this position pAD8 , a mutation is introduced here m pAD9l/92. Similarly, two mutations pAD113 are silent and do not produce an ammo acid change. These silent mutations likely do not contribute substantially to the thermostability of the protein.
EXAMPLE 11: Further thermostabilization of HLADH proteins This example describes the means by which the thermostable proteins identified and characterized as in the prior examples can be further thermostabilized. Using the new mutants as a starting point, the process applied here can be reiterated to increase the thermostability of the HLADH enzyme even further. Namely, it is expected that combinations of the identified HLADH mutations or, combinations of these mutations with other HLADH mutations, can further thermostabilize the enzyme.
In order to do this, the new thermoinactivation limits need to be defined as described in Example 3. This is followed by a new round of mutagenesis performed as described in Examples 8, 9, and 10. In addition, the identified mutations can be put together in differing combinations by in vi tro site-directed mutagenesis and further molecular biology methods (see, e.g., Sambrook et al . , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY. 1989)) that include DNA shuffling via PCR methods (Stemmer et al . , Proc. Natl. Acad. Sci., 91, 10747-10751 (1994a); Stemmer et al . , Nature, 340, 389-391 (1994b)). As they have done in the past, these methods are all expected to give further increases in the levels of thermostability of the enzyme or, in another similarly screened-for trait.
All of the references cited herein, including patents, patent applications, sequences, and publications, are hereby incorporated in their entireties by reference.
While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred embodiments can be used, including variations due to improvements in the art, and that the invention can be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: DAVID C. DEMIRJIAN IGOR A. BRIKUN MALCOLM J. CASADABAN VERONIKA VONSTEIN
(ιi) TITLE OF INVENTION: Method For The Stabilization Of Proteins And The Thermostabilized Alcohol Dehydrogenases Produced Thereby
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(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val lie Lys Cys Lys Ala Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser lie Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg lie Lys Met Val Ala Thr Gly lie Cys Arg 35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60 ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288 Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His H s Phe Leu Gly Thr 130 135 140 AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175 TT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 10 315 GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 2
(l) SEQUENCE CHARACTERISTICS (A) LENGTH 374 amino acids
(B) TYPE amino ac d (D) TOPOLOGY linear
(n) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 2
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60 Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val 65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin 85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Val Asp He
Figure imgf000037_0001
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly
305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350 Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 3
(l) SEQUENCE CHARACTERISTICS (A) LENGTH 1128 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY linear (n) MOLECULE TYPE DNA (genomic)
( i) SEQUENCE DESCRIPTION SEQ ID NO 3 ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro
20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val
50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly
65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro
80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175
TTT TCT ACT GGT TAT GGG CT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG CT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATA AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu He Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GCG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Ala Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 4
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 374 ammo acidr
Figure imgf000039_0001
(D) TOPOLOGY linear
(ill MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 4
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val 65 70 75 80 Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly
180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240 Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
He Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Ala Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly
305 310 315 320 Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met
325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) ΞTRANDEDNEΞS : double (D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96
Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro
20 25 30 AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144
Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val
50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240
He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACC CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro
80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110 TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480
Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528
He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly
160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190 GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GCC ATT GGT CGG 816 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Ala He Gly Arg 260 265 270 CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe
305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 374 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val 65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin 85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He
195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240 Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Ala He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335 Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370 (2) INFORMATION FOR SEQ ID NO .7
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid (C) ΞTRANDEDNESΞ : double
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45 TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AAC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Asn He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288 Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125 AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GCA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220 ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720
He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816
Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300 AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 8
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 374 am o acids (B) TYPE amino acid
(D) TOPOLOGY linear
(ii) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION SEQ ID NO 8
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu
1 5 10 15 Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Asn He Gly Glu Gly Val 65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin
85 90 95 Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn
290 295 300 Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly
305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met
325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe
340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO.9:
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 1128 base pairs (B) TYPE: nucleic acid
(C) ΞTRANDEDNESS : double
(D) TOPOLOGY, linear
(ii) MOLECULE TYPE: DNA (genomic)
(XI ) SEQUENCE DESCRIPTION. SEQ ID NO .9.
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96
Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144
Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60 ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75 GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ATT CCC 288 Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe He Pro 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155 ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315 GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO.10-
(l) SEQUENCE CHARACTERISTICS. (A) LENGTH 374 ammo acids
(B) TYPE ammo acid (D) TOPOLOGY linear (ii) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 10
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30 Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val 65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe He Pro Gin 85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu
260 265 270 Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350 Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 11 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNEΞS : double
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro
80 85 90 95 CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175 TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255 GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC AGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Ser Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GGT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Gly Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350 TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 12
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 374 ammo acids
Figure imgf000048_0001
(D) TOPOLOGY linear
(ii) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 12
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val
65 70 75 80 Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160 Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe
165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Cln Gly
180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240 Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270 Asp Thr Met Val Thr Ala Leu Ser Cys Ser Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Gly Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 13
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 1128 base pairs
(B) TYPE nucleic acid
(C) ΞTRANDEDNEΞΞ double (D) TOPOLOGY linear
(u) MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO 13
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30 AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288 Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110 TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190 GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205 ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATA AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu GCG Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285 GTG AGC GTC ATT GCG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Ala Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365 ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370 (2) INFORMATION FOR SEQ ID NO 14
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 374 ammo acids
Figure imgf000050_0001
(ii) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 14
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser
35 40 45 Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val
65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125 Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
He Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Ala Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335 Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370 (2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESΞ : double
(D) TOPOLOGY, linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15.
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45 TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288 Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC TTC TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ATC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140 AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480
Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys
145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly
160 165 170 175
TTT ACT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576
Phe Thr Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val
195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672
He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220 ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720
He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu
225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr
240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816
Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly
275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300 AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe
320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104
Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 16
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 374 ammo acids (B) TYPE ammo acid
(D) TOPOLOGY linear
(ii) MOLECULE TYPE protein (XI ) SEQUENCE DESCRIPTION SEQ ID NO 16
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60 Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val 65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu
100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Thr Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He
210 215 220 Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys
225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn
290 295 300 Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly
305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 17
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 1128 base pairs (B) TYPE nucleic ac d
(C) ΞTRANDEDNESS double
(D) TOPOLOGY linear
In) MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO 17 ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro
20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCT ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45
TCA GAT GAC CAC GTA GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly 65 70 75 GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC CTC TGC 336 Gin Cys Glv Lys Cys Arg Val Cys Lys H s Pro Glu Gly Asn Leu Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432
Ser Arg Phe Thr Cys Arg Gly Lys Pro He His H s Phe Leu Gly Thr
130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480
Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys
145 150 155 ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528
He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672
He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp
210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA GTC ATT GGT CGG 816 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg 260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315 GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 320 325 330 335 ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO: 18: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE, protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30 Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Ser He Gly Glu Gly Val
65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Thr Pro Gin 85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Leu Cys Leu
100 105 110 Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser
115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val He Gly Arg Leu 260 265 270 Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Val Gly Val Pro Pro Asp Ser Gin Asn Leu Ser Met Asn 290 295 300 Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365
Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO: 19: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 19.
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96 Glu Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro 20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG NNN ACA GGA ATT TGT CGC 144 Lys Ala His Glu Val Arg He Lys Met Val Ala Thr Gly He Cys Arg 35 40 45
TCA GAT GAC CAC NNN GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG NNN ATT GGA GAA GGC 240 He Ala Gly His Glu Ala Ala Gly He Val Glu Xaa He Gly Glu Gly 65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT NNN CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Xaa Pro
80 85 90 95 CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT GAA GGC AAC NNN TGC 336 Gin Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Xaa Cys 100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC ATG CAG GAT GGT ACC 384 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr 115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC CAC TTC CTT GGC ACC 432 Ser Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr 130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG ATC TCA GTG GCC AAG 480 Ser Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys 145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT CTC ATT GGC TGT GGA 528 He Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly 160 165 170 175 TTT NNN ACT GGT TAT GGG TCT GCA GTC AAG GTT GCC AAG GTC ACC CAG 576 Phe Xaa Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin 180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG GGC CTG TCT GTT 624 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG ATC ATT GGG GTG GAC 672 He Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp 210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG GGT GCC ACT GAG 720 He Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG GAG GTG CTG ACA 768 Cys Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr 240 245 250 255
GAA NNN AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA NNN ATT GGT CGG 816 Glu Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa He Gly Arg 260 265 270 CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC NNN CAA GAA GCA TAT GGT 864
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Xaa Gin Glu Ala Tyr Gly 275 280 285
GTG AGC GTC ATT NNN GGA GTA CCT CCT NNN TCC CAA AAT CTC TCT ATG 912 Val Ser Val He Xaa Gly Val Pro Pro Xaa Ser Gin Asn Leu Ser Met 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe 305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe
320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro 340 345 350 TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104 Phe Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128 He Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 374 amino ac-.ds
Figure imgf000057_0001
(ii) MOLECULE TYPE, protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 Ser Thr Ala Gly Lys Val He Lys Cys Lys Ala Ala Val Leu Trp Glu 1 5 10 15
Glu Lys Lys Pro Phe Ser He Glu Glu Val Glu Val Ala Pro Pro Lys 20 25 30
Ala His Glu Val Arg He Lys Met Val Xaa Thr Gly He Cys Arg Ser 35 40 45
Asp Asp His Xaa Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val He 50 55 60
Ala Gly His Glu Ala Ala Gly He Val Glu Xaa He Gly Glu Gly Val 65 70 75 80 Thr Thr Val Arg Pro Gly Asp Lys Val He Pro Leu Phe Xaa Pro Gin
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Xaa Cys Leu 100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gin Asp Gly Thr Ser 115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro He His His Phe Leu Gly Thr Ser 130 135 140
Thr Phe Ser Gin Tyr Thr Val Val Asp Glu He Ser Val Ala Lys He
145 150 155 160 Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu He Gly Cys Gly Phe 165 170 175
Xaa Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gin Gly 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val He 195 200 205 Met Gly Cys Lys Ala Ala Gly Ala Ala Arg He He Gly Val Asp He 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 225 230 235 240
Val Asn Pro Gin Asp Tyr Lys Lys Pro He Gin Glu Val Leu Thr Glu 245 250 255
Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa He Gly Arg Leu 260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Xaa Gin Glu Ala Tyr Gly Val 275 280 285
Ser Val He Xaa Gly Val Pro Pro Xaa Ser Gin Asn Leu Ser Met Asn 290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala He Phe Gly 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu He Thr His Val Leu Pro Phe 340 345 350
Glu Lys He Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser He 355 360 365 Arg Thr He Leu Thr Phe 370
(2) INFORMATION FOR SEQ ID NO 21
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 31 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNEΞΞ single (D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid (xi) SEQUENCE DESCRIPTION SEQ ID NO 21
CCCCGAATTC TCAAAACGTC AGGATGGTAC G 31
(2) INFORMATION FOR SEQ ID NO 22
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 44 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single (D) TOPOLOGY linear
(n) MOLECULE TYPE other nucleic acid
(xi) SEQUENCE DESCRIPTION SEQ ID NO 22
CCCCTCTAGA ATAAATGAGC ACAGCAGGAA AAGTAATAAA ATGC 44

Claims

WHAT IS CLAIMED IS:
1. A method of obtaining a nonnative protein having a thermostability that is increased over that of the native version of said protein, wherein said method comprises :
(a) obtaining a vector a gene that encodes said native protein;
(b) mutating said vector at more than one position m said gene to produce a vector library of cells comprising mutated versions of said gene;
(c) introducing said vector library en masse into cells of a strain in which the majority of said mutated versions of said gene are transcribed and translated to produce a cell library; (d) screening said cell library to identify a cell comprising a mutated version of said gene that encodes a nonnative protein having a thermostability that is increased over that of the wild-type version of said protein; and (e) purifying said cell from said cell library.
2. The method of claim 1 which further comprises isolating from said cell m a vector said mutated version of said gene and, on said mutated version of said gene, repeating steps (b) through (e) .
3. The method of claim 1 wherein said protein is an alcohol dehydrogenase.
4. The method of claim 1 wherein said protein is horse liver alcohol dehydrogenase.
5. The method of claim 1, wherein said screen is carried out in the presence of alcohol.
6. The method of claim 1, wherein said screen is carried out at an increased temperature.
7. The method of claim 1, wherein said strain is either Escherichi coli or Thermus flavus .
8. A method for selecting against growth of Escherichi coli recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Escherichia coli cells, wherein said method comprises growing said recombinant cells under conditions selected from the group consisting of wherein ethanol is present in a concentration of about 10%, isopropanol is present in a concentration of about 4%, and propanol is present m a concentration of about 2%, with the proviso that said wild-type cells exhibit reduced or an absence of growth under said conditions.
9. A method for selecting for growth of Thermus flavus recombinant cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Thermus flavus cells, wherein said method comprises growing said recombinant cells under conditions selected from the group consisting of wherein ethanol is present in a concentration of about 1% in a liquid or solid medium at a pH of about 7.0, and isopropanol is present in a concentration of from about 0.5% to about 1% in a liquid or solid medium at a pH of about 7.0, with the proviso that said wild-type cells exhibit reduced or an absence of growth under said conditions.
10. A method of increasing the thermostability of horse liver alcohol dehydrogenase, which comprises introducing into a gene which encodes said alcohol dehydrogenase a mutation at a codon which codes for an ammo acid residue at a position selected from the group consisting of ammo acid positions 75, 94, 110, 177, 257, 268, 282, 292, and 297.
11. A method of increasing the thermostability of horse liver alcohol dehydrogenase, which comprises changing an ammo acid residue at a position selected from the group consisting of am o acid positions 75, 94, 110, 177, 257, 268, 282, 292, and 297.
12. An isolated and purified nucleic acid comprising a sequence selected from the group consisting of SEQ ID N0:3, SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.
13. An isolated and purified protein comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO : 6 , SEQ ID NO : 8 , SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.
14. A plasmid comprising the nucleic acid sequence of claim 12.
15. A plasmid selected from the group consisting of pAD7, pAD8, pADIO, pAD91, pAD92, pAD93 , pAD95, pADlll, pAD113, and pTG450.
16. A vector library comprising an isolated and purified mixture of vectors comprising mutated versions of a horse liver alcohol dehydrogenase gene.
17. A host cell comprising a plasmid according to claim 14.
18. A host cell comprising a plasmid according to claim 15.
19. A host cell according to claim 17, wherein said cell is a member of the genus of Thermus or Escherichia .
20. A host cell according to claim 18, wherein said cell is strain TGF650.
21. A cell library comprising an isolated and purified mixture of cells obtained by transformation en masse with the vector library of claim 16.
PCT/US1998/009627 1997-05-12 1998-05-12 Method for the stabilization of proteins and the thermostabilized alcohol dehydrogenases produced thereby WO1998051802A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations
CN114517192A (en) * 2020-04-27 2022-05-20 青岛尚德生物技术有限公司 Protease mutant BLAPR1 with improved heat stability and coding gene and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DE BOLLE X ET AL: "Identification of residues potentially involved in the interactions between subunits in yeast alcohol dehydrogenases.", EUR J BIOCHEM, JUL 1 1995, 231 (1) P214-9, GERMANY, XP002076287 *
PARK DH ET AL: "Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.", J BIOL CHEM, MAR 15 1992, 267 (8) P5527-33, UNITED STATES, XP002076288 *
PARK DH ET AL: "isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, 1991, MD US, pages 13296 - 13302, XP002076289 *
RELLOS P ET AL: "Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2.", PROTEIN EXPR PURIF, JUN 1994, 5 (3) P270-7, UNITED STATES, XP002076286 *
WEBER JM ET AL: "A chromosome integration system for stable gene transfer into Thermus flavus.", BIOTECHNOLOGY (N Y), MAR 1995, 13 (3) P271-5, UNITED STATES, XP002076290 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US7037726B2 (en) 2000-05-08 2006-05-02 Genencor International, Inc. Methods for forming recombined nucleic acids
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations
CN114517192A (en) * 2020-04-27 2022-05-20 青岛尚德生物技术有限公司 Protease mutant BLAPR1 with improved heat stability and coding gene and application thereof
CN114517192B (en) * 2020-04-27 2023-06-23 青岛根源生物技术集团有限公司 Protease mutant BLAPR1 with improved thermal stability, and encoding gene and application thereof

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