REARRANGED TISSUE PLASMINOGEN ACTIVATORS AND METHOD FOR PRODUCING SAME
Background of the Invention
This invention relates to the use of recombinant DNA techniques to produce therapeutic proteins, in particular to the use of such techniques to produce novel, modified human uterine tissue plasminogen activator (mtPA) genes and plasmids containing such genes, host cells transformed or transfected thereby, and mtPA molecules produced therefrom.
Tissue plasminogen activator (tPA) is a multi-domain serine protease which catalyzes conversion of plasminogen to plasmin. As such, tPA is of therapeutic value. When administered exogenously, tPA can effect a lysis of blood clots (thrombolysis). tPA has been proven effective in clinical trials for treatment of myocardial infarction. Other indications being examined include pulmonary embolism, deep vein thrombosis and stroke.
The tPA molecule contains five discrete structural domains. In the presence of plasmin, single-chain tPA or zymogen enzyme can be cleaved into an activated two-chain form. The heavy chain contains four of these domains: a "finger" domain which is homologous to a portion of fibronectin; a "growth factor" domain which is homologous to epidermal growth factor; and two non-equivalent "Kringle" domains. Plasmin cleavage to form two-chain tPA occurs C-terminal to Kringle 2 (at Arg27g). The light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin.
tPA i s a relati vely clot-speci fi c plasmi nogen acti vator due to its affinity for fibri n , whi ch forms the clot matrix . Thi s fi bri n
affinity is believed to be due to interactions of the finger and Kringle 2 domains with fibrin. The lower affinity for fibrin by Kringle 1 is not well undestood.
It is one aspect of the present invention to produce an mtPA with higher fibrin specificity and one which can achieve higher rates of fibrinolysis than the wild-type tPA molecule.
It is one aspect of the present invention to provide methods for increasing the spacing between tPA domains for increasing the rate of fibrinolysis or the resistance to inhibition by endogenous tPA inhibitors present in human plasma.
tPA secreted by human melanoma cells was purified and characterized by Rijken fit al- (J- Biol. Chem. 256, 7035 (1981). Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen it al., 3. Clin. Inv. 71, 368 (1983); Koringer ≤t al.. -3- Clin Inv. £2, 573 (1982)). Differences between tPA derived from melanoma and normal uterine tissue have been reported (Pohl £± al. , FEBS Lett. 168, 29 (1984)).
Rijken et al . , Biochem. Biophys. Acta 580, 140 (1979) describes the partial purification, from human uterine tissue, of human tissue plasminogen activator (utPA).
Recombinant DNA techniques have been used previously to obtain mRNA from a line of cancer cells (Bowes melanoma cells), this mRNA being used to produce cDNA encoding Bowes tPA, as described in Goeddel et al . , European Pat. Appln. No. 0093619. Copending, commonly assigned U.S.S.N. 782,686 to Wei et al., fully incorporated herein by reference, describes DNA sequences encoding utPA and further describes site-directed mutagenesis of the DNA sequence at any one or more of the three positions which code for amino acids which in turn normally become glycosylated in post-transl tion processing steps by mammalian
cells. The resultant modified tPA molecules having altered amino acid sequences fail to exhibit glycosylation at the utagenized site. The work has also been reported by Wei et al. , DNA 4, 76 (1985), and in EPA 178,105.
Expression vectors for expression of secreted tPA in mouse cells were subsequently reported by Reddy et al. , J. Cell Biochem. TOP. 154 (1986).
European Patent Application No. 0,234,051 to Pannekoek et al., discuss tPA molecules having rearranged domains but unaltered light chains. Bowes melanoma cells served as source of tPA for the work. It is noted, however, that while the application proports to provide the understanding and tools necessary for designing an actual production of tPA mutants, the description fails to provide a reproducible or predictable method for altering the melanoma cell derived cDNA for providing desired mtPAs.
It is another aspect of the present invention to provide noval methods for predictably insuring the tPA cDNAs are altered in the desired manner to produce the desired mtPAs.
European Patent Application No. 0,231,624 by Marotti et al. , describe other human tissue plasminogen activator analogs having rearranged or deleted native domain regions. The Marotti application describes complex and time consuming procedures for the generation of specified tPA cDNAs by complete chemical synthesis of oligonucleotides. Further, the synthesis was based on native tPA derived from human melanoma cells (Bowes cells).
It is yet another aspect of the present invention to provide simplified, more direct methods for the predictable rearrangement of domains with a tPA like molecule based on utPA.
It is a still further aspect of the present invention to provide unique cDNA sequences encoding tPA like molecules having unique restriction endonuclease sites located at predetermined positions and to provide novel molecules resulting therefrom. 5
It is a further aspect of the present invention to provide novel approaches for generating new molecules having a biological activity associated with tissue plaminogen activator.
0 Summary of the Invention
In accordance with the principles and objects of the present invention there are provided mtPA's which are generated from a parent tPA molecule by deletion, rearrangement or duplication of
15 domains. It was surprising to discover that such alterations still result in molecules having a biological activity associated with w ld- ype or unmodified tPA. It was totally unexpected that some of these novel mtPA's are superior to the parent molecule with respect to their rate of fibrinolysis.
Z0
A preferred method of the present invention comprises introducing at least two restriction enzyme sites into the tPA cDNA. These sites are ideally positioned at the positions in the tPA cDNA corresponding to boundaries of the tPA protein domains
25 although other locations may also serve.
The exact positioning of these sites is critical for preserving desirable activities of the parent tPA molecule. The most preferred embodiments of the present invention have introduced 30 into separate cDNAs an Avr II restriction site, an Nhe I restriction site , an Spe I restriction site , and an Xba I restriction site . The resultant preferred amino acid sequences are listed in Table 3 described below.
35
The altered cDNA's described above can then be advantageously manipulated to generate deletions or duplications of tPA domains as shown in Table 4. These manipulations are described in additional detail below and generally involve cutting the modified cDNA's with the appropriate restriction enzymes and thereafter ligating the resultant cDNA fragments to form new preferred cDNAs.
In the most preferred embodiments, the above modifications have been combined with other modifications for the purpose of obtaining molecules with extended in vivo half-life, for example the conversion of a asparagine to a glutamine at nucleotide 451.
Additional preferred embodiments of the mutant invention include host organisms for maintenance and replication of the sequences, expression vectors for expression of said mtPA's in COS cells, C127 cells, CHO cells, and the mtPA proteins derived from these expression systems.
Brief Description of the Drawings
Further understanding of the principles and objects of the present invention may be had by studying the accompanying Figures wherein:
Figure 1 shows the position of Avr II, Nhe land Spe I sites in the tPA cDNA;
Figure 2 is a schematic diagram of construction of vector expressing mtPA with K, deletion;
Figure 3 is a representation of the LK444BHS vector for transient expression of mtPA;
Figure 4 is a representation of CLH3AXS2DHFR vector for stable expression of mtPA in CHO cells;
Figure 5 is a representation of CLH3AXBPV vector for stable expression of mtPA in C127 cells;
Figure 6 is a schematic diagram of the construction of vector containing mtPA havingK;]^ duplication;
Figures 7a-l and 7a-2 show the construction of the SP.SNA vector
10. containing the Ult rearrangment mtPA sequence; and
Figures 7b-l and 7b-2 show the construction of the SP.ULT containing the ULT rearrangement mtPA sequence.
15
Detailed Description and Best Mode
Definitions
The term "cell culture" refers to the containment of growing cells
Z0f< derived from either a multicellular plant or animal which allows for the cells to remain viable outside the original plant or animal.
The term "host cell" refers to a microorgansim including yeast, bacteria and mammalian cells which can be grown in cell culture and
25 transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expression of such molecule.
The term "domain" refers to a discrete continuous part of an amino
3Q< acid sequence that can be equipped with a particular function. With respect to tPA, references (Banyai, L. et al. , Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator, FEBS Lett. 163(1), 37-41 (1983) and Ny, T. et al. The structure of the Human Tissue-type Plasminogen Activator Gene: Correlation of Intron
35 and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad. Sci. USA 81, 5355-5359 (1984)) have been defined the domain regions and Figure 1 herein discloses the approximate locations of the domain regions.
SUBSTITUTESHEET
The term "downstream" identifies sequences proceeding farther in the direction of expression; for example, the coding region is downstream from the initiation codon.
The term "interdomain" refers to the regions of a protein's amino acid sequence that lie between the domains.
The term "maintained" refers to the stable presence of a plasmid within a transformed host wherein the plasmid is present as an autonomously replicating body or as an integrated portion of the host's genome.
The term "microorganism" includes both single cellular prokaryote and eukaryote organisms such as bacteria actinomycetes and yeast.
The phrase "non-native endonuclease restriction sites" refers to endonuclease restriction sites that are not normally present in the native cDNA and are synthesized at pre-existing restriction sites of the native cDNA sequence.
The term "operon" is a complete unit of gene expression and regulation, including structural genes, regulator genes, and control elements in DNA recognized by regulator gene product.
The term "plasmid" refers to an autonomous self-replicating extrachromosomal circular DNA and includes both the expression and nonexpression types. Where a recombinant microorganism of cell
■ culture provides expression of such molecule is described as hosting an expression plasmid the term "expression plasmid" includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s).
The term "promoter" is a region of DNA involved in binding the RNA polymerase to initiate transcription.
The term "DNA sequence" refers to a single- or double-stranded DNA molecule comprised of nucleotide bases, adenosine, thy idine, cytosine and guanosine and further includes genomic and copy DNA (cDNA).
The term "suitable host" refers to a cell culture or microorganism that is compatible with a recombinant plasmid and w ll permit the plasmid to replicate, to be incorporated into its genome or to be expressed.
The term "upstream" identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream from the coding region.
The term "restriction endonuclease" alternatively referred to as restriction enzymes refers to enzymes which cleave double- stranded DNA (dsDNA) at locations or sites characteristics to the particular enzyme. For example, the restriction endonuclease EcoRl cleaves dsDNA only at locations:
5'GAATTC3' to form 5' G and AATTC3' fragments.
3'CTTAAG5' 3'CTTAA G5'
Although many of such enzymes are known, the most preferred embodiments of the present invention are primarily concerned with only selected restriction enzymes having specified characteristics.
Conventions used to represent plas ids and fragments are meant to be synonymous with conventional representations of plasmids and their fragments.- Unlike the conventional circular figures, the single line figures on the charts represent both circular and linear
double-stranded DNA with initiation or transcription occurring from left to right (5' to 3'). Numbering of nucleotides and amino acids correspond to the particular amino terminal form shown in Table 1, although it will be readily understood the obvious numbering modifications may apply with different NH2 terminal forms. The table below provides the standard abbreviations for amino acids.
Abbreviations for amino acids
General Methods
Methods of DNA preparation, restriction enzyme cleavage, 5 restriction enzyme analysis, gel electrophoresis, DNA fragment isolation, DNA precipitation, DNA fragment ligation, bacterial transformation, bacterial colony selection and grov/th are as detailed in Maniatis et al- , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 1982 (hereafter referred to on 0 Maniatis). Methods of in vitro RNA transcription in a buffered medium and in vitro protein translation in rabbit reticulocyte lysate are as detailed in the manufacturers instructions (Promega Biotech). DNA sequencing was performed using the Sanger dideoxy method using either single-stranded DNA or denatured double-stranded T5 DNA.
Synthetic Oliqonucleotide Linkers
The following oligonucleotide linkers were obtained from zσ: Biolabs Inc.
1. d(CCTCGAGG) 8 raer
2. d(CCCTCGAGGG) 10 mer
3. d(CCCCTCGAGGGG) 12 mer 25
Each linker contains the recognition sequence for the restriciton enzyme Xho I (CTCGAG), which is unique to the tPA cDNA and the SP65 vector. The linkers were utilized in the generation of linker insertion mutants and subsequently for the generation of 30 deletion mutants.
tPA cDNA Source
The cloning of the full-length cDNA of human uterine tPA is 35 described by Reddy et al. (1987). Essentially mRNA was made from
human uterine tissue by the guanidine triocyanate procedure followed by CsCl gradient purification and oligo-dt affinity purification. Reverse transcriptase and Klenow were used to convert the message into double-stranded cDNA which was cloned into the Pst I site of pBR322. The tPA cDNA clone was screened for with oligonucleotides deduced from the sequence of Bowes melanoma tPA. A 2455. base pair cDNA was isolated, sequenced and found to be in good agreement with published sequences (Pennica et al. , 1983). The uterine tPA cDNA differed from melanoma tPA at several sites (predominantly in the 3' untranslated region of the clone) (from Reddy e± al., 1987).
An Sfanl site (nucleotide 16) at the 5' end of the clone near the ATG start codon for tPA and Bglll site (2090) was cleaved, filled in with Klenow in the presence of dNTP's, and Sal I linkers lysated to the blunt ends. The cDNA was recloned into pBR322 as a Sal I fragment and subsequently recloned into other vectors using the Sal I sites.
Generation of a Linker Insertion Mutant
A Spe I linker was placed at the Bgl II (115) site of the tPA cDNA such that it could be used with the other introduced Avr II, Nle I and Spe I sites
A detailed method for the construction of the Bgl II (115) Spe
I (nucleotide 8) mutant is given below.
Iμg of SP6-tPA was cleaved with Bgl II at the unique Bgl II recognition site (nucleotide 115) using the standard protocol. The linearized DNA was precipitated with ethanol and resuspended in nick-translation buffer (40mM KP04 (pH 7.5), 6.6mM M'gCl2, l.OmM mercaptoethanol , 250 μM dATP, dCTP, dTTP and dGTP together with 5μ of DNA polymerase I (Klenow fragment). This procedure fills in the 5' cohesive ends to generate "blunt ended" linearized DNA.
After incubating at room temperature for one hour the Klenow was heat inactivated at 65°C for five minutes. To this mixture was added 100 pmoles of phosphorylated 8 mer Xho I linker (commercially 5 available), ligation buffer (final concentration of 50mM Tris (pH 7.8), lOmM MgCl2, 20mM DTT, I M ATP and 50μg ml" bovine serum albumin) and 200u of T. DNA ligase. The ligation was allowed to proceed overnight at 22°C.
0 The ligated DNA was phenol :chloroform:1AA extracted and ethanol precipitated. This was resuspended in restriction enzyme buffer, overdigested with Xho I and run on a one percent agarose gel to remove multiple linkers and the excess linkers from the relinearized DNA. The relinearized DNA was extracted from the
15 agarose and ethanol precipitated. The precipitated DNA was resuspended in ligation buffer and T. ligase and allowed to ligate overnight at 16°C.
A small al quot of the religated DNA was transfected into the Zϋ. ___ coli bacterial strain DH5 using standard protocols and the transfected bacteria plated on LB agar amp plates.
Bacterial colonies were picked, grown in LB media and DNA prepared on a small scale by standard procedures. The plasmid DNA 25 was analyzed by restriction enzyme analysis and the loss of the unique Bgl II site was confirmed:
Bgl II (115) Spe I (8 mer) 4TSRS5
30 106 126
AGA.GGA.GCC.AGA.TCG.ACT.AGT.CGA.TCT.TAC.CAA arg gly ala arg ser thr ser aro ser tyr gin -1 1 2 3 4 5 6
35
Synthesis of Primers for Site-Specific Mutagenesis
The human uterine tPA cDNA was modified by site-specific mutagenesis using synthetic oligonucleotides prepared by the solid 5 phase phosphotriester method .
The following primers were synthesized and used for such mutagenesis. They are in the anti-sense sequence.
10 1. Primer EGAV
5' GCA.ACT.TTT.CCT.AGG.CAC.TGA 3' for
i ntroduction of an Avr II si te (CCT-AGG) at nucl eoti des 256-261 15
2. Primer SWS
5' GCA.CGT.GGC.ACT.AGT.ATC.TAT.TTC 3' for
20. introduction of a Spe I site (ACT.AGT) at nucleotides 379-384
3. Primer KN1
5' CCCCTGTAACTAGTGCCCTG 3' for 25 introduction of a SPe I site (ACTAGT) at nucleotides 409-444
4. Primer KC1
30 5' GGGTGCTAGCGAACTCTGAG 3' for
introduction of an Nhel (GCTAGC) site at nucleotides 619-624
35
5. Primer KCA1
5 ' GTGCTFCAGCJAGCTGAGCTGTAC 3 ' for
i ntroduction of an Nhe I (GCTAGC) at nucl eoti des 613-618
6. Primer KNB2
5 ' GCCACGGTAGCJAGCCCCATTCCC 3 ' for
i ntroduction of an Nhe I (GCTAGC) si te at nucl eoti des 673-678
7. Primer KC2
5' GTACTCCCIAGGCAGCCTGC 3' for
introduction of an Avr II (CCTAGG) site at nucleotides 871-876
8. Primer KCA2
5' CGTCAGCCTAGGGTTCTTCAGC 3r for
introduction of an Avr II (CCTAGG) site at nucleotides 862-867
9. Primer SWN
5' CAGGCCGCAGCTAGCGCAGGAGGG 3l for
introduction of an Nhe 1 (GCTAGC) at nucleotides 901-906
10. Primer OMT
5' CCCTCCACTAGTGCGAAACTG 3' for
introduction of a SPe I site (ACTAGT) at nucleotides 943-948.
11. Primer Pen
5' CTGGTCACCTAGGCATGTTG 3' for 5 introduction of an Avr II (CCTAGG) at nucleotides 1693-1698
Site Specific Mutagenesis using above Synthesized Oligonucleotides
TO The M13 based oligonucleotide directed mutagenesis procedure is essentially that detailed by Kunkel et al (Proc. NaT'!. Acad. Sci. USA S2, 488 (1985)). Phage containing the M13tPA.MGA vector (the tPA cDNA which has the Asn 451 gin mutation described in the aforementioned copending application and publication of Wei fit al.)
Ϊϊ5 were grown in an E.coli strain CJ236 (which is dut~ ung~) in media in the presence of 0.5 ug ml uridine. The single stranded DNA produced contained uracil residues instead of thymine. The single stranded M13tPA.MGA was prepared by normal procedures.
ZQ 10 ng of the phosphorylated mutating oligonucleotide was annealed with 1 ug of single stranded DNA in a total volume of 20 ul containing 1 x SSC (0.15 M NaCl, 15 mM sodium citrate). The annealing mixture was heated to 70°C then allowed to cool slowly in a water bath for several hours. The annealed fragments were
25 converted in covalently cured circular DNA by the action of DNA polymerase and T4 ligase. The annealed mixture was made up to 100 ul containing 20 mM Hepes (pH7.8), 2 mM DTT, 10 mM MgCl2, 500 uM each of dATP, dTTP, dCTP, and dGTP, ImM ATP, 2.5 units of Klenow and 2 units of T4 DNA ligase. Incubation started at 0°C for 5 minutes,
30 room temperature for 5 minutes, 37°C for 2 hours, and overnight at 16°C.
10 ul of the above reactive mixture was added to 300 ul of competent DH5 bacterial cells and left in ice for 1 hour. The 35 bacteria were heat shocked at 37°C for 5 minutes then serially
diluted into 3 ml of soft agar (0.6%) containing 200 ul of mid-way phase JM101 and poured onto LB agar plates and allowed to solidify. The plates were then incubated overnight at 37βC.
5"- Nitrocellulose lifts were taken from the plates, baked at
32
80°C, under vacuum, for 2 hours and hybridized with the 6 P ATP labeled mutant oligonucleotide. Mutant positive isolates were determined by thermal denaturation of the DNA:oligo duplexes and further grown in OM101 on a larger scale for maxi-prep Rf DNA 10 isolation. The Bam HI-Hind III fragment containing the tPA cDNA including the Asn 451 gin mutation and the new mutation was further recloned into the SP65 vector and/or mammalian cell expression vector using normal procedures of restriction enzyme cleavage and ligation.
I
An example of this method is the generation of mutant KCA2 (R252P;N451Q) in which an Avrll site is introduced into the 3' boundary of the cDNA encoding the Kringle 2 domain.
zα
559 570
AAC.CGC.AGG.CTG
(251) N R R L (254)
25 site-specifi c, ol igonecl eoti de directed mutagenesi s yi el ds
ACC.CCI.AGG.CTG
30 N P R L
Avrll site (CCTAGG).
35 Other mutants generated using this method are as follows:
EGAV (V51R; N451Q)
SWS (R92S; N451Q)
KN1 (I101T; N451Q) KC1 (C171S; N451Q)
KCA1 (E169A; FT70S; N451Q)
KNB2 (S189A; A190S; N451Q)
KC2 (T255P; W256R; N451Q)
KCA2 (R252P; N451Q) SWN (S265A; T266S; N451Q)
OMT (I279T; K280S; N451Q)
Pen (R529P; P530R: N451Q)
(N.B. The unique restriction enzyme sites were introduced with a mutant (MGS (N451Q) hence each contains this mutant also)
The positions of the restriction enzyme sites relative to the domain structure of the tPA protein are as follows (see Figure 1):
Bgl II (115) Spe(8) - between the N-terminus of the mature processed tPA protein and the N-terminus of the finger domain
EGAV - between the C-terminus of the finger domain and the N-terminus of the growth factor domain
SWS - between the C-terminus of the growth factor domain and the
N-terminus of the Kringle 1 domain
KN1 - at the N-terminus of the Kringle 1 domain
KC1 - at the C-terminus of the Kringle 1 domai n
KCA1 - - at the C-terminus of the Kringle 1 domai n
KNB2 - at the N-terminus of the Kringle 2 domai n
KC2 - at the C-terminus of the Kringle 2 domai n
KCA2 - at the C-terminus of the Kringle 2 domai n
SWN - between the C-terminus of the
Kringle 2 domain and the N-terminus of the protease domain
OMT - at the N-terminus of the protease domain
Pen - at the C-terminus of the protease domain
Manipulations of the tPA cDNA:
Avrll, Nhel, Spel and Xbal restriction enzymes recognize unique and separate hexanucleotide sequences ie.
Avrll CCTAGG
Nhel GCTAGC
Spel ACTAGT
Xbal TCTAGA
When cleaved by the appropriate restriction enzyme the 5' overhang is common to all four sites ie.
Avrll
Nhel
Spel
Xbal
Hence fragments with the above ends can be easily ligated and as an extra benefit when the ends from the differently cut sites are ligated the resulting hybrid site is no longer recognized by either restriction enzyme making it diagnostic for the correct ligation and/or orientation of the fragment.
For example
Avrll 5' overhang Nhel 5' overhang
C CTAGC GGATC G i ligate
CCTAGC GGATCG
Since it has lost its palindromic feature it is no longer cleaved by either Avrll or Nhel. The same finding applies to other possible combinations.
Manipulations of the tPA cDNa can be accomplished using the above introduced sites to generate deletions and duplications.
A) Deletions
The Avrll, Nhel and Spel sites located in the following mutants; EGAV, SWS, KNI, KCl, KCAl, KNB2, KC2, KCA2, SWN, OMT and Pen have been utilized to generate deletions in the tPA cDNa which cause a loss of domain(s) in the expressed protein product.
An example of such a manipulation is the deletion of the cDNA encoding the kringle 1 domain using mutants KNI and KCAl.
KNI KCAl
400 420 607 630 GAC-CAG-GGC-ACT-AGT-TAC-AGG AGC-TCA-GCT-AGC-TGC-AGC-ACC-CCT asp gin gly thr ser tyr arg ser ser ala ser cys ser thr pro
98 i 105 167 I 174
Spel cut Nhel cut
1 1 ~ GAC.CAG.GCC.ACT.AG- C.TGC.AGC.ACC.CCT
1 ligate (digest with Spel and Nhel to remove any contaminating KNI or KCAl
400 630
GAC.CAG.GGC.ACT.AGC.TGC.AGC.ACC.CCT asp gin gly thr ser cys ser thr pro
98 99 100 171 172 173 174
ie. Kl del or del (101-170); 100TS171; N451Q
Details of the Kl deletion construction is as follows: 1 μg of plasmid SP6 KNI was digested with Hindlll and Spel and 1 μg of plasmid SP6 KCAl was digested with Hindlll and Nhel. The DNA fragments generated were separated and isolated by gel electrophoresis. The large fragment from SP6 KNI and the small fragment from SP6 KCAl were ligated using normal procedures; redigested with Spel and Nhel and transfected into the E. coli strain DH5 and plated onto LB media plates containing amplicillin. Resistant colonies were picked and grown. Plasmid DNA was prepared on a small scale and analyzed by restriction enzyme analysis for the deletions and loss of the Spel and Nhel sites. The procedure is shown schematically in Figure 2.
The same procedure was used to generate the following series of mutants.
Mutant Lesion Domains Deleted
del (171-190); 170AS191 N451Q Interkringle del (101-170); 100TS171; N451Q Kringle 1 del (189-256); 188AR257; N451Q Kringle 2 del (101-256); 100TR257; N451Q Kringles 1 and 2 del (5-51) 4TR52; N451Q Finger del (51-92) 50S93; N451Q Growth factor del (5-92) 4TS93; N451Q Finger & Growth factor
del (2-266); 1TS267; N451Q The heavy chain. (Finger, growth factor, both kringles)
• In the construction of the mutants encoding the deletions, the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7.
For example, in the generation of the deletion mutant Kldel, a plasmid containing the unique Spe I site (KNI) was ligated with a plasmid containing the unique Nhe I site (KCAl) via their common cohesive ends.
Plasmids and Restriction Enzyme
Mutants Sites Utilized
N del KCAl (Nhel) and KNB2 (Nhel) Kl del KNI (Spel) and KCAl (Nhel) K2 del KNB2 (Nhel) and KCA2 (Avrll) K1K2 del KNI (Spel) and KCA2 (Avrll) F del Bgl/Spe (Spel) and EGAV (Avrll) G del EGAV (Avrll) and SWS (Spel) FG del Bgl/Spe (Spel) and SWS (Spel) Protease Bgl/Spe (Spel) and SWN (Nhel)
B. Duplications
Referring to Figure 1 utilizing the Avrll, Nhel and Spel sites listed and described in the previous sections it was possible to duplicate the DNA encoding domain(s) and hence duplicate the same domain(s) in the protein product. An example of such a duplication is the generation of a tPA analogue containing four kringle domains (a duplication of kringles 1 and 2) using mutants SWS and SWN (see Figure 6).
The following duplications have been constructed in a similar fashion. The amino acid sequences are shown in Tables 4 - 9 and 12 - 15
Mutant Duplicated' Domains
2K1, 1K2 Kringle 1
1K1, 2K2 Kringle 2
2 1, 2 2 Kringles 1 and 2
S+N Kringles 1 and 2
2F Finger
2G Growth factor
2FG Finger and growth factor
2 Prot 1CV Protease domain
2 Prot 2CV Protease domain
In the construction of the mutants encoding the duplications the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7a-l, 7a-2, 7b-l and 7b-2.
For example in the generation of the duplication mutant, 2F, a plasmid containing the introduced unique Avrll (EGAV) was ligated with a plasmid containing the introduced Spel site at the Bglll (115) site (Bgl/Spe) via their common adhesive ends.
Mutant Plasmids and restriction enzymes utilized
EGAV (Avrll) and Bgl/Spe (Spel) SWS (Spel) and EGAV (Avrll) SWS (Spel) and Bgl/Spe (Spel)
KCAl (Nhel) and KNI (Spel) KCA2 (Avrll) and KNB2 (Nhel) KCA2 (Avrll) and KNI (Spel)
SWN (Nhel) and SWS (Spel)
OMT (Spel) and SWS (Spel)
SUBSTITUTESHEET
Mutant Plasmids and restriction enzymes utilized
2 Prot 1CV Pen (Avrll) and OMT (Spel)
2 Prot 2CV Pen (Avrll) and SWN (Nhel)
10 C) Rearrangements
Utilizing the Avrll, Spel and Nhel sites either individually or in combination within a plasmid it was possible to rearrange domains or blocks of domains within the parent molecule such that the final product has a
15 shuffled domain structure.
As an example it was possible to rearrange the tPA cDNA such that the DNA encoding the heavy chain and light chain are switched ie. the the light chain at the amino terminal half of the protein and the heavy chain at the C-terminal half of the protein (see Figures 7a-l, 7a-2, 7b-l and 7b-2).
20
The final product was designated the ult rearrangement mutant.
In a similar manner other mutants were generated with rearranged
25 domains.
2K1 encodes for a tPA analogue in which the kringle 2 domain has been replaced by another kringle 1 domain generating a protein with two kringle 1 domains but no kringle 2. zσ
2K2 encodes for a tPA analogue which contains two kringle 2 domains and no kringle 1 domain.
The amino acid sequences of the rearranged mutants are shown in Tables 10, 11 and 16.
35
SUBSTITUTESHEET
In the construction of the mutants encoding the rearrangements, the following plasmids were appropriately restriction enzyme digested and ligated as in Figures 2, 6 and 7a-l, 7a-2, 7b-l and 7b-2.
For an example, the ULT mutant was generated as in Figures 7a- 1, 7a-2, 7b-l and 7b-2.
10
Plasmids and restriction
Mutant enzymes utilized
15
2K1 KCAl (Nhel), KNI (Spel), KCA2 (Avrll) and KNB2 (Spel)
2K2 KCAl (Nhel), KNI (Spel),, KCA2 (Avrll) and KNB2 (Spel)
ZQ'
ULT Bgl/Spe (Spel), Swn (Nhel) and Pen (Avrll)
25 Verification of mutants
The mutations were verified by restriction enzyme analysis, sequencing and/or in vitro transcription/translation analysis. Mutant encoded proteins with sufficient fibrinolytic activity were analyzed by zymography relative to wild type tPA. 0
Vectors
Sp65-tPA 5
The BamHI-Hindlll fragment containing the tPA cDNA sequence isolated from M13MP18.tPA by restriction enzyme analysis and gel electrophoresis and ligated into the Bam HI, Hindlll cleaved SP65
SUBSTITUTE
vector (Promega Biotech). This orientation (with the 5' end of the cDNA adjacent to the SP6 promoter) enabled an analysis of the mutant protein product by i_n vitro RNA synthesis and i_n vitro protein synthesis. The SP65.tPA vector was also a convenient vector to use during the manipulation of the inserted cDNA e.g. deletion generation.
LK444BHS.tPA
Referring to Figure 3, mutated cDNA molecules were recloned into the LK444BHS vector. The BamHI, Hindlll fragment was isolated from SP65.tPA, a mutant derivative obtained by restriction enzyme cleavage and gel filtration. This fragment was ligated to a BamHI, Hindlll cleavage vector, LK444BHS. This mutation allowed for the transient expression of the tPA analogue in a COS 7 cell line driven by the human β-actin promoter.
CLH3AXBPV.tPA
Referring to Figure 5, the Sal I fragment was isolated from
SP65.tPA, a mutated derivative by restriction enzyme cleavage and gel purification. This fragment was ligated to an Xho I cleaved vector CLH3AXBPV as shown in Figure 5. The orientation was determined and selected such that the inserted sequence was under the driving force of the metallothionine promoter in C127 cells.
CLH3AXSV2DHFR
The tPA cDNA or mutants were restriction enzyme digested with Sail and recloned with the Xhol site cleaved in the vector. The orientation of the tPA cDNA or mutant is such that expression is driven by the metal!othionein promoter. The CLH3AXSV2DHFR vector is used for the stable expression of tPA or modified tPA in CHO cells with the ability for ethatrexate amplification.
Transfection of COS cells
A transient expression system was used wherein the expression vector (LK444BHS) was used to transfect COS-7 cells (ATCC #
CRL1651). Two to three days after introduction of foreign DNA, conditioned medium was analyzed to characterize the activity of the secreted modified tPA protein.
3 x 10 cells were grown in 100 mm plates in DMEM + 10% glutamine for 1 day preceding transfection. Ten to 20 μg of DNA was added to 2.0 ml of tris-buffered saline (pH 7.5). 1 ml of 2 mg/ml DEAE-dextran (made just before transfection by adding 50 mg DEAE-dextran + 25 ml TBS) was added to this solution. Cells were washed 2 times with phosphate-buffered saline (PBS) and the transfection solution added. Cells were incubated at 37°C for 15 -30 minutes. Dextran solution was then removed and cells washed again with PBS 2 times. This solution was replaced with 10 ml DMEM medium (no serum) plus 100 μl chloroquine (10 mM). The cells were then incubated at 37βC for 4 hours. The cells were washed twice with PBS and fed with GIT serum free medium (10 ml).
Transfection of DHFR-CHO Cells
DUKX CHO cells were obtained from Lawrence Chasin of Columbia
University. THese cells are deficient in dihydrofolate reductase.
This gene is present in vector CLH3AXSV2DHFR. Cells were plated in alpha plus media 10% FBS, 1% glutamine medium at a density of 7 x
5 10 cells per 100 mm dish 24 hrs. before transfection. 100-50 μg of plasmid DNA in 0.5 ml transfection buffer (the composition of which is 4 g NaCl , 0.185 g KCl, 0.05 g Na2HP04, 0.5 g dextrose, and 2.5 g HEPES, pH 7.5 per 500 ml total volume). 30 μl of 2M CaCl2 is added to the above solution and the mixture allowed to equilibrate for 45 minutes at room temperature. The medium is removed from the dishes cells washed twice with PBS, and the DNA
solution added to the cells. The cells are allowed to incubate at room temperature for 20 minutes. 5 ml of medium is then added and the cells incubated for four hours at 37βC. The media was removed and the cells were then shocked with 15% glycerol in transfection 5 buffer at 37°C for 3.5 minutes. After 48 hours, the cells were split at a H3 ratio and fed with a selection medium containing 0.02 μM methotrexate. Cell colonies which survive the treatment appear 10 to 14 days after transfection.
O Thereafter, selected colonies were amplified with increasing levels of methotrexate according to published procedures (e.g. Michel et al., Bio/Technology 3, 561 (1985)). Modified tPA proteins produced by these cells was purified by previously reported procedures (Lau et al., Bio/Technology 5, 953 (1987) and U.S.
T5 4,656,134 to Ringold).
Transfection of C127 Cells
Mouse C127 cells were transfected with DNA prepartions ZO according to methods previously published by researchers in
Assignees laboratories (Hsiung et al . , 0. Mol. Appl. Genetics 2, 497 (1984)). Genes encoding modified tPA's were cloned into BPV-based vector CLH3AVBPV and these plasmids used for tranfections.
Z5 Modified tPA proteins were purified from conditioned medium by previously reported procedures (Lau et al . , Bio/Technology 5, 953 (1987)).
Assays of Modi fi ed tPA' s
30
Quanti tation of mtPA protei ns i n condi tioned medi um was performed wi th a commercial ly avai labl e ELISA Ki t for determi nation of tPA from Ameri can Diagnosti ca (Greenwi ch, CT, USA) . The coati ng and detection anti body i s a goat anti -human tPA IgG.
35
Activity was determined by a published spectrophotometric assay for the rate of activation of plasminogen (Verheijen et al . , Thromb. Haemostas. 48, 266 (1982)). The absorbance change ;measured in the assay is converted to Units by reference to a WHO melanoma tPA standard. Specific activity of the mtPA proteins is determined by dividing Units by protein, the latter as determined in the ELISA assay.
Pharmaceutical Applications
10
The mtPAs of the invention may advantageously be admixed with a pharmaceutically acceptable carrier substance, e.g., saline, and administered orally, intravenously, or by injection into affected arteries of the heart. Administration will be generally as is 1.5 carried out for two currently used blood clot lysine enzymes, streptokinase and urokinase.
The mtPA's of the invention may also be used therapeutically to lyse clots in human patients needing treatment of embolisms, 20; e.g., post-operative patients, patients who have recently suffered myocardial infarction resulting in clots, and patients suffering from deep vein thrombi. The following examples are illustrative.
Example 1
25
For emergency treatment of thrombi by bolus injection, 5-1Omg of lyophilized mtPA are mixed together with saline and placed in the chamber of a syringe, which is used to inject the mtPA bolus into the patient intravenously.
30
Example 2
For infusion treatment for the rapid lysis of coronary thrombi, about lOOmg/hr of lyophilized mtPA are infused
35
intravenously over a period of about one hour, followed by intravenous infusion of about 50 mg/hr over a period of about three more hours.
Example 3
For infusion treatment for the rapid lysis of coronary thrombi, the protocol of Example 2 is followed, except that infusion is preceded by the intravenous injection of a bolus of about 10 mg mtPA in saline.
Example 4
For infusion treatment for the slow lysis of deep vein thrombi about 15 mg/hr of lyophilized mtPA dissolved in saline are infused intravenously over a period of about 12-24 hours.
It will now be readily recognized by those skilled in the art that the foregoing amounts are merely representative and are subject to variation depending on the individual characteristics of the particular mtPA selected. It will also be readily apparent that numerous modifications based on the teachings within may be made without departing from the spirit or scope of the present invention, and in particular but without limitation, the mtPAs of the present invention may be used for diagnostic purposes including i_n vitro assays and In vivo imaging application.
T E
TGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA 1 + + + + + + 60
ACACTTCGTTAGTACCTACGTTACTTCTCTCCCGAGACGACACACGACGACGACACACCT
START SIGNAL aa M K R G L C C V L L L C G -
GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGATCT
61 + + + + + + 120
CGTCAGAAGCAAAGCGGGTCGGTCCTTTAGGTACGGGCTAAGTCTTCTCCTCGGTCTAGA PROPEPTIDE «i aa A V F V S P S Q E I H A R F R R G A R S
TACCAAGTGATCTGCAGAGATGAAAAAACGCAGATGATATACCAGCAACATCAGTCATGG
121 + + + + + + 180
ATGGTTCACTAGACGTCTCTACTTTTTTGCGTCTACTATATGGTCGTTGTAGTCAGTACC aa Y Q V I C R D E K T Q M I Y Q Q H Q S W -
CTGCGCCCTGTGCTCAGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCAGGGCA
181 + + + + + + 240
GACGCGGGACACGAGTCTTCGTTGGCCCACCTTATAACGACCACGTTGTCACCGTCCCGT
FINGER DOMAIN aa L R P V L R S N R V E Y C W C N S G R A -
CAGTGCCACTCAGTGCCTGTCAAAAGTTGCAGCGAGCCAAGGTGTTTCAACGGGGGCACC
241 + + + + + + 300
GTCACGGTGAGTCACGGACAGTTTTCAACGTCGCTCGGTTCCACAAAGTTGCCCCCGTGG aa Q C H S V P V K S C S E P .R C F N G G T
TGCCAGCAGGCCCTGTACTTCTCAGATTTCGTGTGCCAGTGCCCCGAAGGATTTGCTGGG
301 + + + + + + 360
ACGGTCGTCCGGGACATGAAGAGTCTAAAGCACACGGTCACGGGGCTTCCTAAACGACCC
GROWTH FACTOR DOMAIN aa C Q Q A L Y F S D F V C Q C P E G F A G
AAGTGCTGTGAAATAGATACCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTACAGG
TTCACGACACTTTATCTATGGTCCCGGTGCΔCGATGCTCCTGGTCCCGTAGTCGATGTCC aa K C C E I D T R A T C Y E D Q G I S Y R - GGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCGTTG CCGTGCACCTCGTGTCGCCTCTCACCGCGGCTCACGTGGTTGACCTTGTCGTCGCGCAAC aa G T W S T A E S G A E C T N W N S S A L
GCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAACCAC
481 + + + + + + 540
CGGGTCTTCGGGATGTCGCCCGCCTCCGGTCTGCGGTAGTCCGACCCGGACCCCTTGGTG
KRINGLE 1 DOMAIN aa A Q K P Y S G R R P D A I R L G L G N H -
AACTACTGCAGAAACCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCGGGG 541 + + + + + +. 600
TTGATGACGTCTTTGGGTCTAGCTCTGAGTTTCGGGACCACGATGCAGAAATTCCGCCCC aa N Y C R N P D R D S K P W C Y V F K A G
AAGTACAGCTCAGAGTTCTGCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGCTAC
601 + + + + + + 660
TTCATGTCGAGTCTCAAGACGTCGTGGGGACGGACGAGACTCCCTTTGTCACTGACGATG aa K Y S S E F C S T P A C S E G N S D C Y
TTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGC
661 + + + +--- + + 720
AAACCCTTACCCAGTCGGATGGCACCGTGCGTGTCGGAGTGGCTCAGCCCACGGAGGACG aa F G N G S A Y R G T H S L T E S G A S C
CTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCC
GAGGGCACCTTAAGGTACTAGGACTATCCGTTCCAAATGTGTCGTGTCTTGGGGTCACGG KRINGLE 2 DOMAIN aa L P W N S M I L I G K V Y T A Q N P S A -
CAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCC
781 + + + + + + 840
GTCCGTGACCCGGACCCGTTTGTATTAATGACGGCCTTAGGACTACCCCTACGGTTCGGG aa Q A L G L G K H N Y C R N P D G D A K P
TGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGC
841 + + + + + + 900
ACCACGGTGCACGACTTCTTGGCGTCCGACTGCACCCTCATGACACTACACGGGAGGACG
«1 aa W C H V L K N R R L T W E Y C D V P S C
TCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTC
901 + + + + + + 960
AGGTGGACGCCGGACTCTGTCATGTCGGTCGGAGTCAAAGCGTAGTTTCCTCCCGAGAAG aa S T C G L R Q Y S Q P Q F R I K G G L F
GCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCC
961 + + + + + + 1020
CGGCTGTAGCGGAGGGTGGGGACCGTCCGACGGTAGAAACGGTTCGTGTCCTCCAGCGGG
PROTEASE DOMAIN aa A D I A S H P W Q A A I F A K H R R S P- -
GGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCC
1021 + + + + + + 1080
CCTCTCGCCAAGGACACGCCCCCGTATGAGTAGTCGAGGACGACCTAAGAGAGACGGCGG aa G E R F L C G G I L I S S C W I L S A A
CACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATAC
1081 + + + + + + 1140
GTGACGAAGGTCCTCTCCAAAGGCGGGGTGGTGGACTGCCACTAGAACCCGTCTTGTATG aa H C F Q E R F P P H H L T V I L G R T Y
CGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAG 1141 + + + + + + 1200
GCCCACCAGGGACCGCTCCTCCTCGTCTTTAAACTTCAGCTTTTTATGTAACAGGTATTC aa R V V P G E E E Q K F E V E K Y I V H K -
GAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCG
1201 + + + + + + 1260
CTTAAGCTACTACTGTGAATGCTGTTACTGTAACGCGACGACGTCGACTTTAGCCTAAGC aa E F D D D T Y D N D I A L L Q L K S D S
TCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTG
1261 + + + + + + 1320
AGGGCGACACGGGTCCTCTCGTCGCACCAGGCGTGACACACGGAAGGGGGCCGCCTGGAC aa S R C A Q E S S V V R T V C L P P A D L
CAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCT
1321 + + + + + + 1380
GTCGACGGCCTGACCTGCCTCACACTCGAGAGGCCGATGCCGTTCGTACTCCGGAACAGA aa Q L P D W T E C E L S G Y G K H E A L S
CCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGC
1381 + + + + + + 1440
GGAAAGATAAGCCTCGCCGACTTCCTCCGAGTACAGTCTGACATGGGTAGGTCGGCGACG aa P F Y S E R L K E A H V R L Y P S S R C
ACATCΔCAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACT
1441 + + + + + + 1500
TGTAGTGTTGTAAATGAATTGTCTTGTCAGTGGCTGTTGTACGACACACGACCTCTGTGA aa T S Q H L L N R T V T D N M L C A G D T -
CGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCC
1501 + + + + + + 1560
GCCTCGCCGCCCGGGGTCCGTTTGAACGTGCTGCGGACGGTCCCGCTAAGCCCTCCGGGG aa R S G G P Q A - L H D A C Q G D S G G P
CTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGC
1561 + + + + + + 1620
GACCACACAGACTTGCTACCGGCGTACTGAAACCACCCGTAGTAGTCGACCCCGGACCCG aa L V C L N D G R M T L V G I I S W G L G
TGTGGACAGAAGGATGTCCCGGGTGTGTACACCAAGGTTACCAACTACCTAGACTGGATT
1621 + + ■-+ + + + 1680
ACACCTGTCTTCCTACAGGGCCCACACATGTGGTTCCAATGGTTGATGGATCTGACCTAA aa C G Q K D V P G V Y T K V T N Y L D W I
CGTGACAACATGCGACCGTGACCAGGAACACCCGACTCCTCAAAAGCAAATGAGATCCCG 1681 + + + + + +
GCACTGTTGTACGCTGGCACTGGTCCTTGTGGGCTGAGGAGTTTTCGTTTACTCTAGGGC aa R D N M R P END
CCTCTTCTTCTTCAGAAGACACTGCAAAGGCGCAGTGCTTCTCTACAGACTTCTCCAGAC 1741 + + + + + + 1800
GGAGAAGAAGAAGTCTTCTGTGACGTTTCCGCGTCACGAAGAGATGTCTGAAGAGGTCTG
CCACCACACCGCAGAAGCGGGACGAGACCCTACAGGAGAGGGAAGAGTGGCATTTTCCCA
1801 + + + + + + 1860
GGTGGTGTGGCGTCTTCGCCCTGCTCTGGGATGTCCTCTCCCTTCTCACCGTAAAAGGGT
GATACTTCCCATTTTGGAAGATTTCAGGACTTGGTCTGATTTCAGGATACTCTGTCAGAT
1861 '■+ + + + + + 1920
CTATGAAGGGTAAAACCTTCTAAAGTCCTGAACCAGACTAAAGTCCTATGAGACAGTCTA
GGGAAGACATGAATGCACACTAGCCTCTCCAGGAATGCCTCCTCCCTGGGCAGAAATGGC
1921 + + + + + + 1980
CCCTTCTGTACTTACGTGTGATCGGAGAGGTCCTTACGGAGGAGGGACCCGTCTTTACCG
CATGCCACCCTGTTTTCAGCTAAAGCCCAACCTCCTGACCTGTCACCGTGAGCAGCTTTG
1981 + + + + + -+ 2040
GTACGGTGGGACAAAAGTCGATTTCGGGTTGGAGGACTGGACAGTGGCACTCGTCGAAAC
GAAACAGGACCACAAAAATGAAAGCATGTCTCAATAGTAAAAGATAACAAGATCTTTCAG
2041 + + + +- + + 2100
CTTTGTCCTGGTGTTTTTACTTTCGTACAGAGTTATCATTTTCTATTGTTCTAGAAAGTC
GAAAGACGGATTGCATTAGAAATAGACAGTATATTTΔTAGTCΔCAAGAGCCCAGCAGCGG
2101 + + + + + + 2160
CTTTCTGCCTAACGTAATCTTTATCTGTCATATAAATATCAGTGTTCTCGGGTCGTCGCC
CTCAAAGTTGGGGCAGGCTGGCTGGCCCGTCATGTTCCTCAAAAGAGCCCTTGACGTCAA
2161 + + + + + + 2220
GAGTTTCAACCCCGTCCGACCGACCGGGCAGTACAAGGAGTTTTCTCGGGAACTGCAGTT
GTCTCCTTCCCCTTTCCCCACTCCCTGGCTCTCAGAAGGTATTCCTTTTGTGTACAGTGT
2221 + + + + + + 2280
CAGAGGAAGGGGAAAGGGGTGAGGGACCGAGAGTCTTCCATAAGGAAAACACATGTCACA
GTAAAGTGTAAATCCTTTTTCTTTATAAACTTTAGAGTAGCATCGAGAGAATTGTATCAT 2281 + + + + + + 2340
CATTTCACATTTAGGAAAAAGAAATATTTGAAATCTCATCGTAGCTCTCTTAACATAGTA TTGAACAACTAGGCTTCAGCAATATTTATAGCAATCCATAGTTAGTTTTTΔCTTTTCGTT
2341 + + + -+ + + 2400
AACTTGTTGATCCGAAGTCGTTATAAATATCGTTAGGTATCAATCAAAΔATGAAAAGCAA GCCACAACCCTGTTTTATACTGTACTTAATAAATTCAGATATATTTTTCACAGTTTTTCC
2401 + + + + + + 2460
CGGTGTTGGGACAAAATATGACΔTGAATTATTTAAGTCTATATAAAAAGTGTCAAAAAGG
TABLE 2
DESIGNATION OF TPA DOMAINS (after Degan et al. 1986)
10
Propeptide/signal 22 to 108 - 29 to - 1
Finger domain 133 to 246 9 to 46
15
Growth factor domain . 268 to 367 54 to 87
Kringle 1 domain 391 to 634 95 to 176
Kringle 2 domain 655 to 900 183 to 264
Protease domain 943 to 1698 279 to 530
Z0<
25
30
TABLE 3
(All non primed letters (eg a)). refer to nucleotide sequences, all pprriimmeedd lleettttiers (eg a )) refer to corresponding amino acid sequences.)
Ayrll. Nhel. Spel. Xbal Mutants
EGAV V51R: N451Q
241 273 a ) CAG.TGC.CAC.TCA.GTG.CCT.AGG.AAA.AGT.TGC.AGC a1) gin cys his ser val pro arg. lys ser cys ser 45 46 47 48 49 50 51 52 53 54 55
SWS R92S: N4510
364 396 b ) TGC.TGT.GAA.ATA.GAT.ACI.AGI.GCC.ACG.TGC.TAC b ) cys cys glu ile asp thr er ala thr cys tyr 86 87 88 89 90 91 92 93 94 95 96
KNI I101T/ N4510
394 423 c ) TAC.GAG.GAC.CAG.GGC.ACT.AGT.TAC.AGG.GGC c ) tyr glu asp gin gly thr ser tyr arg gly
96 97 98 99 100 101 102 103 104 105
KCl C171 S : N4510
607 636 d ) AGC . TCA . GAG . TTC . GCT . AGC . ACC . CCT . GCC . TGC d1 ) ser ser gl u phe al_a ser thr pro al a cys 107 168 169 170 171 172 173 174 175 176
KCAl E169A: F170S: N4510
598 633 e ) GGG.AAG.TAC.AGC.TCA.GCT.AGC.TGC.AGC.ACC.CCT.GCC e1) gly lys tyr ser ser ala er cys ser thr pro ala 164 165 166 167 168 169 170 171 172 173 174 175
KNB2 S189A: A190S: N4510
655 687 f ) TGC.TAC.TTT.GGG.AAT.GGG.GCT.AGC.TAC CGT GGC f ) cys try phe gly asn gly ala ___r tyr arg gly 183 184 185 186 187 188 189 190 191 192 193
KC2 T255P: W256R: N4510
853 888 g ) CTG.AAG.AAC.CGC.AGG.CTG.CCT.AGG.GAG.TAC.TGT.GAT g ) leu lys asn arg arg leu βro arg glu tyr cys asp
249250251 252 253 254255256257 258 259260
KCA2 R252P: N4510
844 879 h ) TGC.CAC.GTG.CTG.AAG.AAC.CCT.AGG.CTG.ACG.TGG.GAG h1) cys his val leu lys asn pro arg leu thr trp glu 246 247 248 249 250 251 252 253 254255 256 257
SWN S265A: T266S: N4510
883 921 i ) TGT.GAT.GTG.CCC.TCC.TGC.GCT.AGC.TGC.GGC.CTG.AGA.CAG i ) ays asp val pro ser cys ala ser cys gly leu arg gin
259 260 261 262 263 264 265 266 267 268 269 270 271
OMT I279T: K280S: N4510
928 963 j ) CAG.CCT.CAG.TTT.CGC.ACT.AGI.GGA.GGG.CTC.TTC.GCC j ) ' gin pro gin phe arg thr fir gly gly leu phe ala 274 275 276 277 278 279 280281 282 283 284 285
PEN R529P: P530R: N4510
1678 1713 k ) ATT.CGT.GAC.AAC.ATG.CCT.AGG.TGA.CCA.GGA.ACA.CCC k ) ile arg asp asn met ___o arg end - 524525 526 527 528 529 530
HEAVY CHAIN DELETIONS
N Del de1(171-190):170AS191:N4510
601 618 679 . 690
1 ) AAG.TAC.AGC.TCA.GAG.TTC.GCLAGC.TAC.CGT.GGC.ACG 1 ) cys tyr ser ser glu phe ala sfir tyr arg gly thr 165 166 167 168 169 170 191 192 193 914
K1 Del del(101-170):100TS171:N4510
394 408 619 633 m ) TAC.GAG.GAC.CAG.GGC.ACT.AGC.TGC.AGC.ACC.CCT.GCC ) tyr glu asp gin gly thr ifir cys ser thr pro ala 96 97 98 99 100 171 172 173 174 175
K∑ Del del(189-256):188AR257:N45TO
658 672 877 891 n ) TAC.TTT.GGG.AAT.GGG.GCLAGG.GAG.TAC.TGT.GAT.GTG n ) tyr phe gly asn gly al_a arg glu try cys asp val 184 185 186 187 188 257 258 259260261
K K2 Del del(101-256):100TR257:N4510
394 408 877 891 o ) TAC.GAG.GAC.CAG.GGC.ACT AGG.GAG.TAC.TGT.GAT.GTG o ) tyr glu asp gin gly thr arg glu try cys asp val 96 97 98 99 100 257 258259260261
F Del del(5-51) ; 4TR52;N451Q
109 120 262 273 p ) GGA.GCC.AGA.TCG.ACT.AGG.AAA.AGT.TGC.AGC p ) gly ala arg ser thr arg lys ser cys ser 1 2 3 4 52 53 54 55
G Del del(51-92);50S93;N45lQ
244 258 285 396 q ) TGC.CAC.TCA.GTG.CCT.AG ♦GCC.ACG.TGC.TAC q ) cys his ser val pro ser ala thr cys tyr 46 47 48 49 50 93 94 95 96
FG Del del(5-92);4TS93;N451Q
109 120 285 396 r ) GGA.GCC.AGA.TCG.ACT.AGT.GCC.ACG.TGC♦TAC r ) gly ala arg ser thr ser ala thr cys tyr -3 -2 -1 1 93 94 95 96
Protease del(2-226);lTS267;N451Q
109 - 120 907 921 s ) GGA.GCC.AGA.TCG.ACT.AGC.TGC♦GGC.CTG.AGA.CAG s ) gly ala arg ser thr ser cys gly leu arg glu -3 -2 -1 1 267 268 269 270 271
2K1 DEL ( 1 90-252 INS ( 102-169)
661 675 412 616 t ) TTT . GGG . AAT . GGG . GCT AGC . TAC . AGG . GGC TAC.AGC. TCA. GCT
865 879
AGG.CTG.ACG.TGG.GAG
t1) phe gly asn gly ala ser tyr arg gly tyr ser ser ala 185 186 187 188 189 102 103 104 105 166 167 168 169
arg leu thr trp gl-u 253 254255256257
2K2 DEL (102-169) INS (190-252)
400 411 676 864 u ) GAC.CAG.GGC.ACT AGC.TAC.CGT.GGC CTG.AAG.AAC.CCT
616 633
ACG.TGC.AGC.ACC.CCT.GCC
u ) asp gly gly thr ser tyr arg gly leu cys asn pro
98 99 100 101 190 191 192 193 249250251 252
ser cys ser thr pro ala 170 171 172 173 174 175
TABLE 4
MUTANT 2F
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPRSRSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEID TRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV FKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLG KHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKH RRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDT YDNDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYP SSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDV PGVYTKVTNYLDWIRDNMRP
,
TABLE 5
MUTANT 2G
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQ GISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSE FCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPD GDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFL CGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQ LKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHL LNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTN YLDWIRDNMRP
TABLE 6
Mutant 2FG
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTSRSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVP VKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWN SSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSA YRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQER FPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLP PADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
TABLE 7
MUTANT 2K1 1K2
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY TO FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSASTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMIL IGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGG LFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEE QKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEA T5 LSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGR MTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
∑σ
25
3:0
5
TABLE 8
MUTANT 1K1 2K2
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNPSYRGTHSLTESGASCLPWNSMILIGKVY TAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADI ASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEV EKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFY SERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVG IISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
TABLE 9
MUTANT 2K1 2K2
TO
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNPSYRGTWSTAESGAECTNWNSSALAQKPY
T5 SGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTE SGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGL RQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVI LGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDW TECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQG
ZO DSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
Z5
30
35
TABLE 10
MUTANT 2K1
O
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGASYRGTWSTAESGAECTNWNSS ALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSARLTWEYCDVPSCSTCGLRQYSQP
T QFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYR WPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELS GYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPL VCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
ZO
Z5
30
35
TABLE 11
MUTANT ZKZ
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGTSYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALG LGKHNYCRNPDGDAKPWCHVLKNPSCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGK VYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFA DIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKF EVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSP FYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTL VGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP
TABLE 12
MUTANT S+N
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCASATCYEDQGISYRGT WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPAC SEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWC HVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILIS SCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSR CAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTD NMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRD NMRP
TABLE 13
MUTANT OMS
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQA FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIR LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWN ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFR ATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV AGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLG NYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKH SPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDT NDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYP RCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDV VYTKVTNYLDWIRDNMRP
TABLE 14
MUTANT 2 Prot 1 CV
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALY FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIK GGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGE EEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKH EALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLND GRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSGGLFADIASHPWQAAIFAKHRRSPGERFLC GGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQL KSDSSRCAQESSWRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLL NRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNY LDWIRDNMRP
TABLE 15
MUTANT 2 Prot 2 CV
GARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQAL FSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRL LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNS ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRI GGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPG EEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGK EALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLN GRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSCGLRQYSQPQFRIKGGLFADIASHPWQAAI FAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRWPGEEEQKFEVEKYIVHKE DDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAH RLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGC QKDVPGVYTKVTNYLDWIRDNMRP
TABLE 16
MUTANT Ult
GARSTSCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQER FPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSWRTVCLP PADLQLPDWTECELSGYGKHEALSPFYSERLKEAHBRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMPSRSYQVICRDE KTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGF AGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDR DSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNP SAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCAR
TABLE 17
Mutant Specific Activity (mlU/ug)
MSI 350
KNI * 485
10 KCl * 584
KCAl 517
KNB2* 418
KC2 745
KCA2* 617
15
Ndel v.l. Kl del 120 K2del 400 K1 K2del v.l.
20~
2K1, 1K2 320 1K1, 2k2 50 2K1, 2K2 v.l.
25 where v.l. = very low activity and * signifies the most preferred molecules
30
35