CA2290074A1 - Method for the stabilization of proteins and the thermostabilized alcohol dehydrogenases produced thereby - Google Patents

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 SUBSTITUTE SHEET (RULE 26) 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", _In Enzymes as Catalysts in Or anic 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, 1- (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 ~3-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 SUBSTITUTE SHEET (RULE 26) r. i r 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 nonrefrigerated 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 implementa~.ion 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 in 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., "Crosslinked enzyme crystals as robust biocatalysts", Proceedings of the Materials Research Society 1993 Symposium, Biomolecular Materials by Design (1993)), SUBSTITUTE SHEET (fiULE 26) i i i 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 SUBSTITUTE SHEET (RULE 26) r. , ~

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

5 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 vitro 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 pTG100kan'=z 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:1]
that encodes the HLADH protein [SEQ ID N0:2], with the SUBSTITUTE SHEET (RULE 26) i i i 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 vitro 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 SUBSTITUTE SHEET (RULE 26) SEQ ID N0:2). Notably, with respect to SEQ ID N0:2, this protein does not initiate with methionine (Met).
However, other varients of horse liver alcohol dehydrogenase produced by in vitro 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
N0:2. Namely, the second position of the aforementioned protein would be equivalent to the first position of the protein of SEQ ID N0:2. 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;
SUBSTITUTE SHEET (RULE 26) (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 Thermos.
SUBSTITUTE SHEET (RULE 26j ~r i r WO 98/51802 PCT/US98/09b27 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, S 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 Thermos, and desirably is of the species Thermos flavus, Thermos aqua ticus, Thermos thermophilus, or Thermos sp.
Optimally a cell is either an Escherichia coli cell or a Thermos 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 Thermos or Eshcerichia cell. It also is preferable that a vector have an ability to shuttle between different cells, e.g., between a Thermos 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 SUBSTITUTE SHEET (RULE 26) 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 5 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 10 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 SUBSTITUTE SHEET (RULE 26) ,r i T

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 in the method of the invention are set out in 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.
SU8ST1TUTE SHEET (RULE 26) i i i 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 traits) 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 Eschericia 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 40, 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 SUBSTITUTE SHEET (RULE 26) cells which comprise levels of alcohol dehydrogenase that are higher than those of wild-type Thermus flaws cells. This method comprises growing the recombinant cells under conditions selected from the group consisting of wherein ethanol is present at a concentration of aboutlo 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 N0:4, SEQ
ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID
N0:14, SEQ ID N0:16, SEQ ID N0:18 and SEQ ID N0:20. 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
N0:3; SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID NO:15, SEQ ID N0:17 and SEQ
ID N0:19.
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;
pAD$, pADlO, pAD9l, 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.
SUBSTITUTE SHEET (RULE 26) 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 ~l of assay buffer (83 mM KH2P04 [pH
7.31, 40 mM KCl, 0.25 mM EDTA), and sonicated. The assay ~ll~-~T~TtIT~ ~~T ~yJt~~ 2~~
,fi , , IS
mixture contains 500 ~.l of cell extract, 100 ~1 EtOH, 20 ~.1 100 mM NAD, 830 ~1 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 identity 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. cola.
This example describes a plate screen for ADH
activity that can be employed, for instance, in E. cola.
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 bisulfate to 400 ml batches of precooled (45oC) Luria agar. Most of the dye is immediately converted to the leuco form by reaction with bisulfate to produce a rope-colored medium. Ethanol diffuses into the E. cola 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.
SUBSTITUTE SHEET (RULE 26) i i 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 IS gene encoding the HLADH enzyme was plasmid pBPP (Park et al., J. Biol. Chem., 266, 13296-13302 (1991)).
E. coli DHS~, cells containing plasmid pBPP (i.e., HLADH') or plasmid pCRII ( i . a . , 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 in Buffer 1 (10 mM KMes, pH 6.5, 0.5 mM
CoCl2, 0.1% Triton X-100, 50 ~Cg/ml lysozyme, 10 ~Cg/ml DNAse) in a chloroform bath for about one hour, washed once in Buffer 2 (10 mM KMes, 0.5 mM CoCl2, 0.2% BSA), and then washed two more times in Buffer 3 (Buffer 2 without BSA). The filters were then incubated at high temperatures in Buffer 4 (10 mM glycine, 0.5 mM CoCl2) 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 phenazine methosulfate, 1 mg/ml nitroblue tetrazolium) at room temperature for 3-5 minutes.
Results of these experiments are depicted in Figure 2. As can be seen in this figure, the experiments confirm that a 15-20 minute treatment of the filters at 75oC resulted in roughly 90% inactivation of the HLADH
SUBSTITUTE SHEET (RULE 26) r. i T

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-rosaniline assay for verification of the activity of the HLADH gene in Thermus In order to allow expression of the HLADH gene in both Thermus and E. coli, the gene was subcloned into the Thermus shuttle vector, pTGl00kantr2 to create plasmid pTG450 depicted in Figure 3. In this construct, the gene is placed upstream of the thermostable kanamycin resistance gene (kantr2) which is commanded by the lac promoter in E. coli, and the leu promoter in Thermus.
An E. coli strain harboring pTG450 has three times more HLADH activity in the presence of IPTG than the strain harboring the original pBPP plasmid. When transformed into Thermus, the adh gene integrates into the leuB site in the Thermus chromosome by a double recombination event. For these experiments, Thermus flavus was transformed with both the HLADH plasmid pTG100kantr2 (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-rosaniline 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 Luria broth.
A standard p-rosaniline plate can not be used since the indicator dye will spontaneously convert to the Schiff base if incubated overnight in the plate as part of this assay.
SUBSTITUTE SHEET (RULE 26) 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. co~i strains harboring the adh gene.
For these experiments, E. coli DH5a cells containing either pTG100kan'r2 (i.e., HLADH-) or pTG450 (i.e., HLADH+) were grown on LB plates with different alcohols in concentrations ranging from 2% to 120. The results of one such experiment are displayed in Table 1.
SUBSTITUTE SHEET (RULE 26) i T

N ~ i r-1 I
(a I i i Cl~ m O
W I i i W

t i 'r-i d' N t t r~l W

O
U rcS

N
'-1 i i U ~ p ' ~-I m w-i O
v ~

fa U O p ~ ~ +

U1 ~ i U H
(,i~ t i ~
W t t dP N t t O O

U ~ ~ ~ i ~~i w U O ~

~ i U7 C ' t O ~o m + +

U

U ~ + + t U ~r + +

tLS w +
.~ O I + +
~-I

J, oW j N t +
~r1 t N +
+ + O

td .,..i o +
~ '_., ~ + + p p O

f.a U

Z$ .~ m + +

N
+ + v ~r + + .d w I + + p N t t W

W y7 v ~1 O

I V

H ' ! a w Wn SUB STITUTE SHEET (RULE
26) As can be seen from Table l, E. coli cells harboring high activity of HLADH (i.e., transformed with the HLADH'plasmid pTG450) are more sensitive to the 5 presence of the alcohols in high concentrations. This probably is due to the accumulation of toxic aldehyde levels in the cells which result from the alcohol dehydrogenase reaction. Three other alcohols were tested (i.e., benzyl alcohol, hexyl alcohol, and hexyl 10 amine), but did not give clear results because of their poor solubility in the media.
The experiment was repeated several times and the alcohol levels were refined to determine a range resulting in a clear selection. Three of the alcohols, 15 i.e., ethanol at a concentration of 100, 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 20 scheme can be employed for the isolation of mutants with altered ADH activity and, in 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 in Thermos This example describes the growth of Thermos strains in the presence of the high concentrations of SUBSTITUTE SHEET (RULE 26) ,~ i ~

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, l, 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.
SUBSTITUTE SHEET (RULE 26}

n-1 N O N

O

C o 0 O ~ N N

x O N

f3~O

m ~n o~

op o ~

N r1 N

O O

rW
p r-1 m1 O r-i ~ e-i r c0 x G1~W tf1 N 01 dP O N ~-1 V' N r-I

r-I

O N rl ri O ~ O O

O

1a r1 Wp x c~a~ 0 0 0 2f H

N ~ N Q1 op O O ,-i N

O

f~

N t"T

N

O O

ra ~p r-1 r-I

O o (~ L lt1 10 N

x o b ri ~D
N

i U ~ N N

L

C1.

N

41 m1 u1 ro x ~
~ x ~

H v N

SUBSTITUTE SHEET (RULE 26) 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 in 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: Hydroxylamine mutagenesis of the adh gene.
This example describes mutagenesis of the adh gene as a representative alcohol dehydrogenase gene using the mutagen hydroxylamine (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 ~.g of plasmid DNA was mixed with 0.5 M NHZOH 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 DH5a, and plated onto LBAPioo Plates ( i . a . LB
plates containing 100 ~g/ml ampicillin). Transformants SUBSTITUTE SHEET (RULE 26) i i were analyzed by the ADH filter assay described in Example 3, and also using the p-rosaniline assay described in 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 inactivation 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 in Example 3. For heat inactivation'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 XL1 containing the LADH' plasmid pBPP, and strain NM554 containing the LADH plasmid pBluescript).
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 in enzyme thermostabilization might be further increased by excising the mutagenized gene from the vector, and resubcloning 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.
SUBSTITUTE SHEET (RULE 26) 'T I 1 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
5 ID N0:21]
CCC CTC TAG AAT AAA TGA GCA CAG CAG GAA AAG TAA TAA AAT
GC
ADH(XbaI) [SEQ ID N0:22]
The adh gene was amplified using these primers and cloned 10 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., su ra) in which the limiting dNTP
15 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.
20 Mutagenized adh-containing fragments were digested using XbaI 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 25 into E. coli strain NM554 to allow the adh gene to be transcribed from the Iac 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 in Table 3.
SUBSTITUTE SHEET (RULE 26) 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.l was done according to Spee et al., su ra, (i.e. with 14 ~.M of limiting dNTP and 200 ~M dITP) and Method No. 2 was done according to Rellos et al., supra (i.e. without dITP and with 25 ~.M 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, pADlO, pAD9l, 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 SUBSTITUTE SHEET (RULE 26) W ~ 1 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.
S
EXAMPLE 9: Characterization of thermotolerant HLADH candidates.
This example describes a characterization for increased thermostability o~ 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 in appropriate media, and cell extracts were made by sonication. The extracts were then incubated at 70°C, taking an initial sample (to), 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 to activity (residual activity) in order to compare the individual samples to each other and adjust for variations in expression levels or growth variations.
Figure 4 displays the residual activity data for the nine candidate plasmids pAD7, pADB, pADlO, pAD9l, pAD92, pAD93, pAD95, pADlll, and pAD113, wherein the to 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 pAD9l, pAD92, and pADlO showed the most noticeable alterations SUBSTITUTE SHEET (RULE 26) 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 ~g/ml Amp (12.5 ug/ml Tet for WT
clone) overnight, concentrated in 1 ml of the assay buffer (83 mM KHZPO4, 40 mM KC1, 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 e~-r~;n R~r 15 min 30 min 60 min pADH7 8 100% 4 50% 2 25% 0.6 8%

pADHB 21 100% 7.4 35% 2 10% 0.2 1%

pADHlO 16 100% 4 25% 1.4 9% 0 0%

pADH91 11 100% 8 73% 6 55% 4 36%

pADH92 25 100% 15 60% 17 68% 12 48%

pA.DH93 6 100% 1 17% 2.5 42% 0 0%

pADH95 66 100% 21 32% 10 15% 3 5%

pADHlll 22 100% 15 68% 16 73% 11 50%

pADH113 9 100% 4 44% 3 33% 0.8 9%

WT 10 100% 1 10% 0.3 3% 0 0%

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 ~g/ml Amp (12.5 ~g/ml Tet for WT clone) overnight, concentrated in 50 ml of the assay buffer (83 mM KHzP04, 40 mM KC1, 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.
SUBSTITUTE SHEET (RULE 26) Table 5 HLADH Substrate Specificity Strain Ethanol Isopropanol Butanol eenzyl Alcol pADH7 8.7 0 5.3 1.4 pADHB 18.2 1.4 11 7 pADHlO 15.6 3 11.5 4.7 pADH91 13.2 1.1 4.7 3.4 pADH92 23.5 2.3 11 6.8 pADH93 5.6 1 3 1.6 pADH95 48 0.7 21.3 4.5 pADH111 22.6 1.6 9.8 3 pADH113 7 1.1 3 5 WT 9.2 1.7 7.6 3.5 Strain Hexanol Cvclohexanol R-(-)Butanol S-(+)Butanol ~ADH7 4 3 0 0 pADH8 _l5 49 2.4 2.2 pADHlO 15 69 10 4 pADH91 5.8 23 2.4 1.7 pADH92 10.6 50 2.3 2.4 pADH93 3.9 22 2 1.4 pADH95 21.3 16.5 0.5 0.8 pADHlll 9.4 58 4 2.7 pADH113 2.7 14.7 2 1.3 WT 10 42 4.3 2.9 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.
SUBSTITUTE SHEET (RULE 26) Table 6. Mutations identified in thermotolerant candidates Mutant Base Amino Original Mutant Amino plasmid pair acid codon codon acid position position' change2 pAD7 774 257 ATG ATA Met257I1e 878 292 GTG GCG Va1292A1a pAD8 285 94 ACT ACC no as change 806 268 GTC GCC Va1268A1a pADlO 227 75 AGC AAC Ser75Asn pAD91/92 284 94 ACT ATT Thr94Ile pAD93 847 282 TGT AGT Cys282Ser 893 297 GAT GGT Asp297G1y pAD95 774 257 ATG ATA Met257I1e 878 292 GTG GCG Va1292A1a pADlll 532 177 TCT ACT Ser177Thr pAD113 129 42 GCC GCT no as change 159 52 GTG GTA no as change 331 110 TTC CTC Phe110Leu Also, the individual sequences of the mutant adh 5 sequences are set forth in the Sequence Listing for pAD7 (i.e., nucleic acid sequence at SEQ ID N0:3 and amino acid sequence at SEQ ID N0:4), pAD8 (i.e., nucleic acid sequence at SEQ ID N0:5 and amino acid sequence at SEQ ID
N0:6), pADlO (i.e., nucleic acid sequence at SEQ ID N0:7 10 and amino acid sequence at SEQ ID N0:8), pAD91/pAD92 (i.e., nucleic acid sequence at SEQ ID N0:9 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 N0:12), pAD95 (i.e., nucleic acid sequence at SEQ ID
15 N0:13 and amino acid sequence at SEQ ID N0:14), pADlll SUBSTITUTE SHEET (RULE 26) ~ T

(i.e., nucleic acid sequence at SEQ ID N0:15 and amino acid sequence at SEQ ID N0:16), and pAD113(i.e., nucleic acid sequence at SEQ ID N0:17 and amino acid sequence at SEQ ID N0:18).
The first numbered amino acid in the wild-type and mutant sequences is serine since, in the sequences studied, the initial methionine (Met) is not present in the final protein. However, it is possible that Met is present in the wild-type (or mutant) HLADH sequences that are produced in a different host, e.g., in 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 pAD9l, PAD92, pAD93, and pAD95 were identified from the same filter and mutants containing plasmids pADll1 and pAD113 were identified from the same filter assay. Also, in both pADB and pAD91/92, the coding sequence specifying amino acid 94 is mutated. Whereas this results in no change in this position in pAD8, a mutation is introduced here in pAD91/92. Similarly, two mutations in pAD113 are silent and do not produce an amino 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 SUBSTITUTE SHEET (RULE 26) i i 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 vitro 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.
SUBSTITUTE SHEET (RULE 26) m i r SEQUENCE LISTING
(1) GENERAL
INFORMATION:

S

(i) APPLICANT: DAVID C. DEMIRJIAN

IGOR A. BRIKUN

MALCOLM J. CASADAHAN

VERONIKA VONSTEIN

to (ii) TITLE OF INVENTION: Method Proteins For The Stabilization Of And The Thertnostabilized Alcohol ehydroge nases ProducedThereby D

(iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Mcdonald Berghoff Boehnen Hulbert &

(B) STREET: 300 South blacker Drive (C) CITY: Chicago 20 (D) STATE: Illinois (E) COUNTRY: United States (F) ZIP: 60606 (v) COMPUTER READABLE FORM:

ZS (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 3 (vi) CURRENT APPLICATION DATA:
O

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(2) INFORMATION
FOR
SEQ
ID
NO:1:

(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:

AGC AAA TGC AAA GCG

S Ser Thr Ala Gly Lys Val Ile AlaValLeuTrp ~ Lys Cys Lys Ala GAA GAG GAG GTG GAG

Glu Lys Lys Pro Phe Ser Ile ValAlaProPro Glu Glu Glu Val Glu SS 2p 25 30 GCC ATG GTG GCC ACA

Lys His Glu Val Arg Ile Lys GlyIleCysArg Ala Met Val Ala Thr GAT ACC CTT GTC ACA

Ser Asp His Val Val Ser Gly ProLeuProVal Asp Thr Leu Val Thr C)S ATC GGC CAT GAG GCA GCG GGC ATTGGAGAAGGC 240 GCA ATT GTG GAG AGC

Ile Gly His Glu Ala Ala Gly IleGlyGluGly Ala Ile Val Glu Ser 65 '10 '15 ACT AAA GTC ATC CCA

Val Thr Val Arg Pro Gly Asp LeuPheThrPro Thr Lys Val Ile Pro gp 85 90 95 TGT AAG CAC CCT GAA

Gln Gly Lys Cys Arg Val Cys GlyAsnPheCys Cys Lys His Pro Glu AAA CGG GGA ACC ATG

Leu Asn Asp Leu Ser Met Pro GlnAspGlyThr Lys Arg Gly Thr Met AGG CCC ATC CAC CAC ACC

Ser Phe Thr Cys Arg Gly Lys PheLeuGly Arg Pro Ile His His Thr SUBSTITUTE SHEET (RULE 26) i i i ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly GCC AAG GTC ACC CAG

Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln GTG GGC CTG TCT GTT

Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 1S 19s zoo 2os ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala G1y Ala Ala Arg Ile Ile Gly Val Asp GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr GAA GTC ATT GGT CGG

3 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe 0 Glu Val Ile Gly Arg CAA GAA GCA TAT GGT

Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly 3S 27s 2so zes CAA AAT CTC TCT ATG

Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met AAA GGA GCT ATT TTT

Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe CTT GTG GCC GAT TTT

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe ACC CAT GTT TTA CCT

S Met Ala Lys Lys Phe Ala Leu Asp Pro Leu I1e 0 Thr His Val Leu Pro CGC TCT GGA GAG AGT

Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

6S (A) LENGTH: 374 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ,70 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu ,7S1 5 10 15 Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser g0 35 40 45 Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile SUBSTITUTE SHEET (RULE 26) ~ T

Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln .S 85 90 95 Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 10 Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser I Jr 130 135 140 Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys ~eu Ile Gly Cys Gly Phe 20 lss 170 17s Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly 25 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg !le Ile Gly Val Asp Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys Val Asn Pro Gln Asp Tyr Lys Lys pro Ile Gln Glu Val Leu Thr Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe SS Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2) INFORMATION FOR SEQ ID N0:3:
(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 N0:3:

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp g 0 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg SUBSTITUTE SHEET (RULE 26) i i i ACA CCT CTT CCT GTG

Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly CCA CTC TTT ACT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys ATG CAG GAT GGT ACC

ZO Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr CAC TTC CTT GGC ACC

Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr ?.S130 135 140 ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys O CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly S GCC AAG GTC ACC CAG

Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln GTG GGC CTG TCT GTT

4O Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp G~S210 215 220 GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu O CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr S GAA GTC ATT GGT CGG

Glu Ile Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg CAA GAA GCA TAT GGT

6O Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly CAA AAT CTC TCT ATG

Val Ser Val Ile Ala Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met AAA GGA GCT ATT TTT

Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe O CTT GTG GCC GAT TTT

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe ACC CAT GTT TTA CCT

Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro CGC TCT GGA GAG AGT

Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile P.rg Thr Ile Leu Thr Phe SUBSTITUTE SHEET (RULE 26) 'I I 1 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acid:.
(B) TYPE: amino acid to (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
IS Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu 1 s to is Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val 3 0 Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu 35 loo los 110 Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile 45 Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys 60Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu Ile SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal Ser ValIleAlaGlyValProProAspSerGlnAsnLeuSerMetAsn Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly 75Gly PheLysSerLysAapSerValProLysLeuValAlaAspPheMet Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle Arg ThrIleLeuThrPhe SUBSTITUTE SHEET (RULE 26) i i i (2) INFORMATION FOR SEQ ID NO: S:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GCG GCT GTG CTG TGG

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp ZO

GAG GTT GCA CCC CCG

Glu Glu Lys Lys Pro Phe Ser Ile G1u Glu Val Glu Val Ala Pro Pro ?.S AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144 ACA GGA ATT TGT CGC

Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg ACA CCT CTT CCT GTG

3 Ser Asp Asp His Val Val Ser Gly Thr Leu Val ~ Thr Pro Leu Pro Val AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 3S 6s 70 7s CCA CTC TTT ACC CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro g0 85 90 95 GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys ATG CAG GAT GGT ACC

Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr CAC TTC CTT GGC ACC

Sfl Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys SS 14s lso lss CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly GCC AAG GTC ACC CAG

Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln C)S GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624 GTG GGC CTG TCT GTT

Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala Gly Ala A1a Arg Ile Ile Gly Val Asp GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr go GAA GCC ATT GGT CGG

Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Ala Ile Gly Arg SUBSTITUTE SHEET (RULE 26) Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln G1u A1a Tyr Gly Val Ser Val ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe 3 O (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids (B) TYPE: amino acid 3 5 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys 4S zo zs 30 Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser S O Asp Asp His Val val Ser Gly Thr Leu Val Thr Pro Leu Pro val Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val SS
Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu loo los llo Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser 6S Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile g O Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys SUBSTITUTE SHEET (RULE 26) i i i Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu S Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Ala Ile Gly Arg Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 20 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe 3 (2) INFORMATION FOR SEQ ID N0:7:
O

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1128 base pairs (8) TYPE: nucleic acid 35 (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi1 SEQUENCE DESCRIPTION: SEQ ID N0:7:

GCG GCT GTG CTG TGG

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp GAG GTT GCA CCC CCG

Glu Glu Lys Lys Fro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro S
O

~G GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144 ACA GGA ATT TGT CGC

Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg S ACA CCT CTT CCT GTG

Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val AAC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Asn Ile Gly Glu Gly CCA CTC TTT ACT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys ATG CAG GAT GGT ACC

Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr CAC TTC CTT GGC ACC

Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr ATC TCA GTG GCC AAG

80 Ser Thr Phe Ser Gln Tyr Thr Val Va1 Asp Glu Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly SUBSTITUTE SHEET (RULE 25) ( i AAG

PheSerThrGly GlySerAlaValLysValAlaLysValThrGln i8o 185 190 GlySerThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerVal 1~

IleMetGlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAsp 210 215 22p IleAsnLysAspLysPheAlaLysAlaLysGluValGlyA1aThrGlu 20 CysValAsnProGlnAspTyrLysLysProIleGlnGluValLeuThr GluMetSerAsnGlyGlyValAspPheSerPheGluValIleGlyArg 25 zso 265 270 LeuAspThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGly ValSerValIleValGlyValProProAspSerGlnAsnLeuSerMet AsnProMetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePhe 40 GlyGlyPheLysSerLysAspSerValProLysLeuValAlaAspPhe MetAlaLysLysPheAlaLeuAspProLeuIleThrHisValLeuPro PheGluLysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSer IleArgThrIleLeuThrPhe SS

(2)INFORMAT IONFORSEQID
N0:8:

( i) EQUENCE CHARACTE RISTICS:
S

(A)LENGTH: 374aminocids a (B)TYPE:minoacid a (D)TOPOLOGY: inear l (i i) TYPE: otein MOLECULE pr 6S (x i) EQUENCE DESCRIPT ION:SEQ N0:8:
S ID

SerThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu 70 GluLysLysProPheSerIleGluGluValGluValAlaProProLys AlaHisGluValArgIleLysMetValAlaThrGlyIleCysArgSer 75 35 40 q5 AspAspHisValValSerGlyThrLeuValThrProLeuProValIle AlaGlyHisGluAlaAlaGlyIleValGluAsnIleGlyGluGlyVal ThrThrValArgProGlyAspLysValIleProLeuPheThrProGln SUBSTITUTE SHEET (RULE 26) Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer Thr PheSerGlnTyrThrValValAspGluIleSerValAlaLysIle Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrGlnGly Ser ThrCysAIaValPheGlyLeuGlyGlyValGlyLeuSerValIle Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal 275 2eo 28s Ser ValIleValGlyValProProAspSerGlnAsnLeuSerMetAsn Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle Arg ThrIleLeuThrPhe (2) INFORMATION FOR SEQ ID N0:9:
(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 N0:9:

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val SUBSTITUTE SHEET (RULE 2fi) 'f I T

AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly CCA CTC TTT ATT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Ile Pro GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys ATG CAG GAT GGT ACC

Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr 1 Met Gln Asp Gly Thr CAC TTC CTT GGC ACC

Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln GTG GGC CTG TCT GTT

Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly 3S Val Gly Leu Ser Val ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp 40 21o zls z2o GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr S GAA GTC ATT GGT CGG

Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg CAA GAA GCA TAT GGT

Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly CAA AAT CTC TCT ATG

Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met AAA GGA GCT ATT TTT

Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe CTT GTG GCC GAT TTT

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro CGC TCT GGA GAG AGT

Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Zle Leu Thr Phe (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26) (A) acids LENGTH:

amino (B ) amino id TYPE: ac (D ) linear TOPOLOGY:

S ( ii)MOLECULETYPE: rotein p ( xi)SEQUENCEDESCRIPTION : Q NO:10:
SEID

Ser ThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu O

Glu LysLysProPheSerIleGluGluValGluValAlaProProLys IS Ala HisGluValArgIleLysMetValAlaThrGlyIleCysArgSer Asp AspHisValValSerGlyThrLeuValThrProLeuProValIle 20 so ss 60 Ala GlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal 65 70 7s 80 Thr ThrValArgProGlyAspLysValIleProLeuPheIleProGln 25 es 90 9s Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu 3 Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer O

Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer Thr PheSerGlnTyrThrValValAspGlu_TleSerValAlaLysIle Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe 40 16s 170 17s Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrGlnGly 45 Ser ThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerValIle 195 200 20s Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu 55 245 250 25s Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu 60 Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal Ser ValIleValGlyValProProAspSerGlnAsnLeuSerMetAsn Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe 75 Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle Arg ThrIleLeuThrPhe (2) INFORMATION FOR SEQ ZD NO:11:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26) r ~ t (A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) IO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GCG GCT GTG CTG TGG

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp GAG GTT GCA CCC CCG

Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro l i Lys A
a H
s Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg ACA CCT CTT CCT GTG

Ser Asp Asp His Val Val Ser Gly Thr Leu Val 2S Thr Pro Leu Pro Val o s ss so AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Giu Ser Ile Gly Glu Gly 30 65 7 7s CCA CTC TTT ACT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro S GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys L
L

eu ys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr AGC AGG TTC ACC T'GC AGA GGG AAG CCC ATC CAC 432 CAC TTC CTT GGC ACC

Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His 4S His Phe Leu Gly Thr ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly GCC AAG GTC ACC CAG

Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gl S
h y er T
r Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg 6S Ile Ile Gly Val Asp GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr GAA GTC ATT GGT CGG

Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg g0 CAA GAA GCA TAT GGT
A
Th sp Leu r Met Val Thr Ala Leu Ser Cys Ser Gln Glu Ala Tyr Gly CAA AAT CTC TCT ATG

Val Ser Val Ile Val Gly Val Pro Pro Gly Ser G1n Asn Leu Ser Met SUBSTITUTE SHEET (RULE 26) Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu val Ala Asp Phe l0 320 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2) INFORMATION FORSEQID
N0:12:

(i)SEQUENCE CHARACTERISTICS:

(A)LENGTH :

amino acids (B)TYPE: amino acid (D)TOPOLOGY:
linear O

(ii) TYPE:
MOLECULE protein (xi) DESCRIPTION: SEQ N0:12:
SEQUENCE ID

35 Ser ThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu Glu LysLysProPheSerIleGluGluValGluValAlaProProLys Ala HisGluValArgIleLysMetValAlaThrGlyIleCysArgSer Asp AspHisValValSerGlyThrLeuValThrProLeuProValIle Ala GlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal Thr ThrValArgProGlyAspLysValIleProLeuPheThrProGln Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer Thr PheSerGlnTyrThrValValAspGluIleSerValAlaLysIle 65 Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrClnGly Ser ThrCysAlaValPheG1yLeuGlyGlyValGlyLeuSerValIle Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys 80 Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu SUBSTITUTE SHEET (RULE 26) r m Asp ThrMetValThrAlaLeuSerCysSerGlnGluAlaTyrGlyVal S Ser ValIleValGlyValProProGlySerGlnAsnLeuSerMetAsn Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet Ala LysLysPheAlaLeuAspProLeuileThrHisValLeuProPhe S

Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle Z0 Arg ThrIleLeuThrPhe 2 S (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double 3 0 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 3 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
S

GCG GCT GTG CTG TGG

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp GAG GTT GCA CCC CCG

Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro ACA GGA ATT TGT CGC

Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg S ACA CCT CTT CCT GTG

Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu SS Ser Ile Gly Glu Gly 6s CCA CTC TTT ACT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro GAA GGC AAC TTC TGC

Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys C)STTG AAA AAT GAT CTG AGC ATG CCT CGG rGA ACC 389 ATG CAG GAT GGT ACC

Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr h h Ser Arg P
e T
r Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu 7S Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly g0 160 165 170 175 GCC AAG GTC ACC CAG

Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln SUBSTITUTE SHEET (RULE 26) GGC CTC TCT GTT

Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val ATC ATT GGG GTG GAC

I1e Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr ~S 240 245 250 255 GTC ATT GGT CGG

Glu GCG Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg CAA GAA GCA TAT GGT

Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly CAA AAT CTC TCT ATG

Val Ser Val ile Ala Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met GGA GCT ATT TTT

3 O Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe GTG GCC GAT TTT

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe CAT GTT TTA CCT

Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro CGC TCT GGA GAG AGT

Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe SO (2) INFORMATION FOR SEQ ID N0:14 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids (B) TYPE: amino acid SS (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi1 SEQUENCE DESCRIPTION: SEQ ID N0:14:
6O Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser 70 Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val 7S Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln gO Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu loo los zlo Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser SUBSTITUTE SHEET (RULE 26) I I T

Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe IO Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile IS Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Iie Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu ZS Ile Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal O

Ser ValIleAlaGlyValProProAspSerGlnAsnLeuSerMetAsn Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet 4O Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle Arg ThrIleLeuThrPhe SO (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid SS (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 6O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 8O Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro SUBSTITUTE SHEET (RULE 26) i SO

Gln CysGlyLysCysArgValCysLysHisProGluGlyAsnPheCys TTG AAAAATGATCTGAGCATGCCTCGGGGAA~CATGCAGGATGGTACC 384 Leu LysAsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThr lls 120 lzs Ser ArgPheThrCysArgGlyLysProIleHisHisPheLeuGlyThr Ser ThrPheSerGlnTyrThrValValAspGluIleSerValAlaLys Ile AspAlaAlaSerProLeuGluLysValCysLeuIleGlyCysGly Phe ThrThrGlyTyrGlySerAlaValLysValAlaLysValThrGln Gly SerThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerVal Ile MetGlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAsp S

Ile AsnLysAspLysPheAlaLysAlaLysGluValGlyAlaThrGlu Cys ValAsnProGlnAspTyrLysLysProIleGlnGluValLeuThr Glu MetSerAsnGlyGlyValAspPheSerPheGluValIleGlyArg Leu AspThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGly SO

Val SerValIleValGlyValProProAspSerGlnAsnLeuSerMet Asn ProMetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePhe 60 Gly GlyPheLysSerLysAspSerValProLysLeuValAlaAspPhe Met AlaLysLysPheAlaLeuAspProLeuIleThrHisValLeuPro Phe GluLysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSer ,70 Ile ArgThrIleLeuThrPhe (2) INFORMATION FORSEQID
N0:16:

(i)SEQUENCE CHARACTERISTICS :

(A)LENGTH : 4 37amino acids g (B)TYPE:
0 amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: rotein p SUBSTITUTE SHEET (RULE 26) (xi) DESCRIPTION: SEQIDN0:16:
SEQUENCE

SerThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu GluLysLysProPheSerIleGluGluValGluValAlaProProLys AlaHisGluValArgIleLysMetValAlaThrGlyIleCysArgSer AspAspHisValValSerGlyThrLeuValThrProLeuProValIle IS AlaGlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal ThrThrValArgProGlyAspLysValIleProLeuPheThrProGln 2o CysGlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser Azg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser 3 d Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe 35 Thr Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Va1 Thz Gln Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile 4 5 Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val GIy Ala Thr Glu Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu 5o Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val 55 z75 2so zss Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 65 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) i i (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

GCG GCT GTG CTG TGG

Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp GAG GTT GCA CCC CCG

Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro ACA GGA ATT TGT CGC

Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg ACA CCT CTT CCT GTG

Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val AGC ATT GGA GAA GGC

Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly CCA CTC TTT ACT CCC

Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro GAA GGC AAC CTC TGC

3 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro 0 Glu Gly Asn Leu Cys ATG CAG GAT GGT ACC

Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr 3S lls lzo 12s CAC TTC CTT GGC ACC

Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu G1y Thr ATC TCA GTG GCC AAG

Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys CTC ATT GGC TGT GGA

Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly GCC AAG GTC ACC CAG

S Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val 0 Ala Lys Val Thr Gln GTG GGC CTG TCT GTT

Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val ATC ATT GGG GTG GAC

Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr GAA GTC ATT GGT CGG

70 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg CAA GAA GCA TAT GGT

Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly CAA AAT CTC TCT ATG

Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met AAA GGA GCT ATT TTT

Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe SUBSTITUTE SHEET (RULE 26) ~ T

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro IO Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2) INFORMATION FOR SEQ ID N0:18:
2 O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 25 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
3 O Sei Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys 35 Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln 45 85 9p g5 Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Leu Cys Leu 5 O Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly 65 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys G1u Va1 Gly Ala Thr Glu Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu 75 z4s z5o zss Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu gO Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn SUBSTITUTE SHEET (RULE 26) i i ProMetLeuLeuLeuSerGlyArgThrTrpLysGly IlePheGly Ala S GlyPheLysSerLysAspSerValProLysLeuVal AspPheMet Ala AlaLysLysPheAlaLeuAspProLeuIleThrHis LeuProPhe Val GluLysIleAsnGluGlyPheAspLeuLeuArgSer GluSerIle Gly ArgThrIleLeuThrPhe S

(2) N0:19:
INFORMATION
FOR
SEQ
ID

Z (i)SEQUENCE
O CHARACTERISTICS:

(A) pairs LENGTH:

base (B) nucleic acid TYPE:

(C) double STRANDEDNESS:

(D) linear TOPOLOGY:

ZS

(ii)MOLECULE DNA(genomi c) TYPE:

(Xi)SEQUENCE SEQID :
DESCRIPTION: N0:19 O

GGA

SerThrAla LysValIleLysCysLysAlaAlaValLeuTrp Gly S CCA

Glu GluLysLys PheSerIleGluGluValGluValAlaProPro Pro GTC

40 Lys AlaHisGlu ArgIleLysMetValAlaThrGlyIleCysArg Val NNN

Ser AspAspHis ValSerGlyThrLeuValThrProLeuProVal Val GAG

Ile AlaGlyHis AlaAlaGlyIleValGluXaaIleGlyGluGly Glu 6s 70 75 SO

AGA

Val ThrThrVal ProGlyAspLysValIleProLeuPheXaaPro Arg S TGC

Gln CysGlyLys ArgValCysLysHisProGluGlyAsnXaaCys Cys CTG

60 Leu LysAsnAsp SerMetProArgGlyThrMetGlnAspGlyThr Leu TGC

Ser ArgPheThr ArgGlyLysProIleHisHisPheLeuGlyThr Cys CAG

Ser ThrPheSer TyrThrValValAspGluIleSerValAlaLys Gln TCA

Ile AspAlaAla ProLeuGluLysValCysLeuIleGlyCysGly Ser TAT

Phe XaaThrGly GlySerAlaValLysValAlaLysValThrGln Tyr GCC

g0 Gly SerThrCys ValPheGlyLeuGlyGlyValGlyLeuSerVal Ala AAA

Ile MetGlyCys AlaAlaGlyAlaAlaArgIleIleGlyValAsp Lys SUBSTITUTE SHEET (RULE 26) ~r i t SS

GTG GGT GCC ACT GAG

Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu S Val Gly Ala Thr Glu CAG GAG GTG CTG ACA

Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr l0 240 245 250 255 GAA NNN ATT GGT CGG

Glu Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa Ile Gly Arg CAA GAA GCA TAT GGT

Leu Asp Thr Met Val Thr Ala Leu Ser Cys Xaa Gln Glu Ala Tyr Gly Val Ser Val Ile Xaa Gly Val Pro Pro Xaa Ser Gln Asn Leu Ser Met AAA GGA GCT ATT TTT

Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp ZS Lys Gly Ala Ile Phe CTT GTG GCC GAT TTT

Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe O

ACC CAT GTT TTA CCT

Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro S CGC TCT GGA GAG AGT

Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe (2)INFORMATION SEQ ID N0:20:
FOR

(i)SEQUENCECHARACTERISTICS:

(A) adds LENGTH:

amino (B) TYPE:
amino acid (D) TOPOLOGY:
linear O

( ii)MOLECULETYPE: protein (xi) SEQUENCEDESCRIPTION: N0:20:
SEQ ID

S SerThrAlaGly Val Ile LysA1aAlaValLeuTrpGlu S Lys Lys Cys GluLysLysPro Ser Ile ValGluValAlaProProLys Phe Glu Glu AlaHisGluVal Ile Lys XaaThrGlyIleCysArgSer Arg Met Val AspAspHisXaa Ser Gly ValThrProLeuProValIle Val Thr Leu 6S So Ss 60 AlaGlyHisGlu Ala Gly GluXaaIleGlyGluGlyVal Ala Ile Val 70 ThrThrValArg Gly Asp IleProLeuPheXaaproGln Pro Lys Val Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Xaa Cys Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser g0 130 135 140 Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile SUBSTITUTE SHEET (RULE 26) i i Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Xaa Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile l~ Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys 15 Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa Ile Gly Arg Leu Asp Thr Met Val Thr Ala Leu Ser Cys Xaa Gln Glu Ala Tyr Gly Val S Ser Val Ile Xaa Gly Val Pro Pro Xaa Ser Gln Asn Leu Ser Met Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly 3 O Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met 3 5 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile Arg Thr Ile Leu Thr Phe 4S (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single S ~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid SS (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

6O (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (7S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:

SUBSTITUTE SHEET (RULE 26)

Claims (21)

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 in a vector a gene that encodes said native protein;
(b) mutating said vector at more than one position in 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 in 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 in 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 amino acid residue at a position selected from the group consisting of amino 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 amino acid residue at a position selected from the group consisting of amino 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 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.

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, pAD10, pAD91, pAD92, pAD93, pAD95, pAD111, 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.