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Title:
MODIFIED GENE SEQUENCES ENCODING MODIFIED TPA AND PRODUCTS THEREFROM
Document Type and Number:
WIPO Patent Application WO/1989/007145
Kind Code:
A1
Abstract:
Novel modified DNA sequences are provided encoding for modified tissue plasminogen molecules. The sequences are modified by insertion of restriction site containing linkers into pre-existing restriction sites in the tPA DNA. The inserted restriction sites are unique to the tPA DNA thereby allowing for predictable deletions, modifications, and spacing changes in the tPA amino acid sequence.

Inventors:
LIVINGSTON DAVID J (US)
MARKLAND WILLIAM (US)
Application Number:
PCT/US1989/000464
Publication Date:
August 10, 1989
Filing Date:
February 03, 1989
Export Citation:
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Assignee:
INTEGRATED GENETICS INC (US)
International Classes:
C12N15/09; C07K14/00; C12N5/10; C12N9/64; C12N9/72; (IPC1-7): C12N15/00; C07H21/04; C12N5/00
Foreign References:
EP0231624A11987-08-12
Other References:
Protein Engineering '87, Abstracts, 5-8 April 1987, The Examination Schools University Oxford,(GB) E.F. Rehberg et al.: "A cassette gene for tissue plasminogen activator: a model for studying protein domain funtion", see page 238, abstract 46
The EMBO Journal, vol. 7, no. 9, 25 August 1988, IRL Press Ltd. Oxford (GB) M.J. Gething et al.: "Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain", pages 2731-2740
Proc. Natl. Acad. Sic, USA, vol. 83, no. 13, July 1986 Biochemistry, Washington, DC (US) A. J. van Zonneveld et al.: "Autonomous functions of strcutural domains on human tissue-type plasminogen activator" pages 4670-4676,
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Claims:
Claims
1. What is claimed is: DNA sequence comprising the sequence: 126 AGA.AGA.GGA.GCC.AGA.TCC.CTC.GAG.GGA.TCT.TAC.CAA and all sequences which hybridize thereto under stringent conditions.
2. DNA sequence comprising the sequence: 288 TGC.AGC.GA .CCA.AGC.CTC.GAG.GCA.AG .TGT.TTC and all sequences which hybridize thereto under stringent conditions.
3. DNA sequence comprising the sequence: 540 AGG.CCA.GAC.GCC.ATC.CCT.CGA.GGG.CTG.GGG.AAC.CAC « and all sequences which hybridize thereto under stringent conditions.
4. DNA sequence comprising the sequence: 1215 GTC.CAT.AAG.GAA.TTC.CTC.GAG.GAA.TTC.GAT.GAT.GAC and all sequences which hybridize thereto under stringent conditions.
5. DNA sequence comprising the sequence: 1359 GAC.TGG.ACG.GAG.TGT.GCC.CTC.GAG.GGC.TCC.GGC.TAC and all sequences which hybridize thereto under stringent conditions.
6. 6 DNA sequence comprising the sequence: 1665 CCG.GGT.GTG.TAC.ACC.AAG.CCT.CGA.GGC.AAG.GTT.ACC.AAC and all sequences which hybridize thereto under stringent conditions.
7. DNA sequence comprising the sequence: 126 AGA.GGA.GCC.AGA.TCG.ACT.AGT.CGA.TCT.TAC.CAA and all sequences which hybridize thereto under stringent conditions.
8. DNA sequence comprising the sequence: 120 280 288 AGA.GGA.GCC.AGA.TCC.CTC.GAG.GCA.AGG.TGT.TTC and all sequences which hybridize thereto under stringent conditions.
9. DNA sequence comprising the sequence: 120 539 543 AGA.GGA.GCC.AGA.TCC.CCC.CTC.GAG.GGG.CTG.GGG.AAC.CAC.AAC and all sequences which hybridize thereto under stringent conditions.
10. DNA sequence comprising the sequence: 120 730 741 AGA.GGA.GCC.AGA.TCC.CCT.GCA.GGG.AAT.TCC.ATG.ATC and all sequences which hybridize thereto under stringent conditions.
11. DNA sequence comprising the sequence: 120 883 894 AGA.GGA.GCC.AGA.TCC.CTC.GAG.GAC.TGT.GAT.GTG.CCC and all sequences which hybridize thereto under stringent cond tions.
12. DNA sequence comprising the sequence: 281 539 553 TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.CTG.GGG.AAC.CAC.AAC and all sequences which hybridize thereto under stringent conditions.
13. DNA sequence comprising the sequence: 279 730 741 TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.AAT.TCC.ATG.ATC and all sequences which hybridize thereto under stringent conditions.
14. DNA sequence comprising the sequence: 279 883 891 TGC.AGC.GAG.CCA.AGC.CTC.GAG.GAC.TGT.GAT.GTG and all sequences which hybridize thereto under stringent conditions.
15. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 1.
16. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 2.
17. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 3.
18. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 4.
19. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 5.
20. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 6.
21. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 7.
22. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 8.
23. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 9.
24. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 10.
25. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 11.
26. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 12.
27. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 13.
28. The product produced by a host cell transformed or transfected with an expression vector comprising the sequence of Claim 14.
29. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 1.
30. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 2.
31. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 3.
32. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 4.
33. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 5.
34. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 6.
35. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 7.
36. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 8.
37. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 9.
38. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 10.
39. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 11.
40. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 12.
41. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 13.
42. A host cell transformed or transfected with a vector comprising the DNA sequence of Claim 14.
43. A method for providing an altered DNA sequence encoding a molecule having a tPA biological property, said sequence capable of being rearranged in a predetermined manner comprising the steps of: a) providing a DNA sequence encoding a molecule having a tPA biological activity; and b) inserting at at least two restriction sites normally present in said sequence a linker containing at least one unique restriction site to form a modified DNA sequence and wherein said linker has a mer length selected to ensure that correct translational reading frame is maintained.
44. The method as provided in claim 43 further comprising the steps of: a) cleaving said modified DNA sequence at at least two of said unique restriction sites to form fragments; and b) ligating said fragments together.
45. The method as provided in claim 44 wherein said normal restriction sites are selected from the group of nucleotide positions consisting of 115, 277, 515, 1201, 1345 and 1652.
46. The method as provided in claim 45 wherein said unique restriction site is Xhol.
Description:
MODIFIED GENE SEQUENCES ENCODING MODIFIED TPA AND PRODUCTS THEREFROM

Background of the Invention

This invention relates to the use of reco binant 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 tPA 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 (thro bolysis). 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 Arg 275 ). The light chain contains the serine protease domain, which is homologous to trypsin and chymotrypsin.

tPA is a relatively, clot-specific plasminogen activator due to its affinity for fibrin, in turn responsible for forming the clot matrix. This fibrin affinity s bel eved to be due to interact i ons of the finger and Kringle 2 domains with fibrin. The participation of other domains in fibrin interactions is not well understood.

Inter domain effects are also poorly understood, do partially to the lack of a three-dimensional structure for tPA.

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 et al. (J. Biol. Chem. 256, 7035 (1981). Therapeutic utility of exogenous tPA was demonstrated with the melanoma-derived material (Collen et al. , J. Clin. Inv. 71, 368 (1983); Korninger £t al., 3. Clin Inv. 69, 573 (1982)). Differences between tPA derived from melanoma and normal uterine tissue have been reported (Pohl et 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 RNA 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-translation processing steps by mammalian

cells. The resultant modified tPA molecules having altered amino acid sequences, fail to exhibit glycosylation at the mutagenized 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. , 3. Cell Biochem. 10D, 154 (1986).

The utility of recombinant wild-type tPA as a human therapeutic is somewhat limited due to the large dose required (Verstraete §£ al. , Lancet 1, 842 (1985)). and may be accompanied by an unacceptably high incidence of bleeding complications due to non clot-specific activation of plasminogen (Verstraete ei al. , J. Pharm. Exp. Ther. 235, 506 (1985)). Additional undesirable properties of wild-type tPA include its short in vi o half-life which can be lengthened by alteration of glycosylation of the protein by site directed mutagenesis ,(Lau et al. , Bio/Technology 5, 953 (1987), fully incorporated herein by reference.

It is an aspect of the present invention to engineer new mtPA possessing improvement of the fibrin affinity and catalytic ability, most preferably, in combination with other improved characteristics.

European Patent Application No. 0,234,051 to Pannekoek ≤± 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 purports to provid the understanding and tools necessary for designing and 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 novel methods for predictably insuring the tPA cDNAs are altered in the desired manner to produce the desired mtPAs.

European Patent Appl cation No. 0,231,624 by Marotti ~t 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 o 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 noval molecules resulting therefrom.

It is a further aspect of the present invention to provide novel approaches for generating new molecules having a biological activity associated with tissue plasminogen activator.

Summary of the Invention

In accordance wi th the principl es and objects of the present i nvention there are provided improved mtPA' s generated by addi tions or del etions of ami no acids from the parent tPA mol ecul e at preexi sti ng retri ction sites . Surprisi ngly, some of these mtPA' s di splay substantial ly improved properti es over wi Id-type tPA with respect to fibrin affinity, fibri nolysis and resi stance to human pl asma tPA i nhi bi tors . Al so provided are DNA sequences modifi ed to contai n uni que restri ction si tes whi ch do not occur

elsewhere in the cDNA and do not occur in the cDNA of mature or wild-type tPA. These modified cDNA's are then advantageously manipulated to generate DNA sequences with partial deletions of domains or deletions of entire domains. Vectors for expression of these sequences in mammalian cells are disclosed along with characterization of the resultant mtPA proteins.

The most preferred embodiments of the present invention include cDNA's coding for utPA comprising one or more of the following modifications: Xho I restriction endonuclease site inserted at position 115, Xho I site inserted at position 277, Xho site inserted at position 515, Xho I site inserted at position 729, Xho I site inserted at position 879, Xho I site inserted at positio 1201, Xho I site inserted at position 1345, Xho I site inserted at position 1652, and Spe I site inserted at position 115 where the position numbers are from the nucleotide sequence. It is important to recognize that thes manipulations may directly lead to either insertions or deletions of amino acids in the resultant sequences, and may have the effect of altering spacing between the domains of the tPA protein. The results of such alterations cannot be predicted. Also described herein are cDNA's derived from those listed above where sequence corresponding to nucleotide bases between Xho I sites in one mtPA and another mtPA have been deleted. In one preferred embodiment the mtPA with Xho I site at position 11 and the mtPA with Xho I site at position 277 has been used to construct a cDNA with nucleotides 121-276 deleted. Other deletion mutant embodiments have been similarly constructed and are detailed below.

Additional preferred embodiments of the instant invention include host organisms for maintenance and replication of the sequences. Still other preferred embodiments include 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 invention may be had by reference to the figures wherein:

Figure 1 shows utPA wild-type amino acid sequence in a two-dimensional representation showing location of domains and of restriction enzyme insertion sites;

Figure 2 depicts plasmid construction showing deletion of the

DNA encoding the finger domain;

Figure 3 shows the expression vector LK 444 BHS used for transient expression of modified tPA;

Figure 4 shows a vector for stable expression of mtPAs in CHO cells; and

Figure 5 shows a vector for stable expression of mtPAs in C127 cells.

Detailed Description and Best Mode

Definitons

The term "cell culture" refers to the containment of growing cells 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 transfected or transformed with a plasmid or vector containing a gene encoding a molecule having a tPA biological characteristic and expressing such molecule.

The term "domain" refers to a discrete continuous part of an amino acid sequence that can be associated with a particular function. With respect to tPA, suitable references describing the domain regions include (Banyai, L. ei & , 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 and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad.

Sci. USA 81, 5355-5359 (1984)). Table 2 illustrates the locations of the domain regions as used herein.

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, yeast, and mammalian cells.

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. When a recombinant microorganism or cell culture providing expression of a 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, thymidine, 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 will 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 s upstream from the coding region.

The term "restriction endonuclease", alternatively referred t herein as a restr ction enzyme refers to one of a class of 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' fragmen 3'CTTAAG5' 3'CTTAA G5'

Although many such enzymes are known, the most preferred embodiment of the present inventions are primarily concerned with only selecte restriction enzymes having specified characteristics.

All cited references are fully incorporated herein by reference.

Conventions used to represent plasmids and fragments are mean 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 linea 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 that obvious numbering modifications will apply to molecules with different NH 2 terminal forms. The table below provides the standard conventional abbreviations for amino acids as they are used herein.

Abbreviations for amino acids

Three-letter One-lette

Amino Acid abbreviation symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Asparagine or aspartic acid Asx B

C

Q E Z G H I L K

M F P S T

W Y V

General Methods

Methods of DNA preparation, restriction enzyme cleavage, restriction enzyme analysis, gel electrophoresis, DNA fragment isolation, DNA precipitation, DNA fragment ugation, bacterial transformation, bacterial colony selection and growth are as detailed in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 1982 (hereafter referred to as 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-strande DNA.

Synthetic Oliqonucleotide Linkers

The following oligonucleotide linkers were obtained from Biolabs Inc.

1. d(CCTCGAGG) 8 mer

2. d(CCCTCGAGGG) 10 mer

3. d(CCCCTCGAGGGG) 12 mer

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 o linker insertion mutants and subsequently for the generation of deletion mutants.

tPA cDNA Source

The cloning of the full-length cDNA of human uterine tPA is described by Reddy §£ l. (1987). Essentially mRNA was made from human uterine tissue by the guanidine thiocyanate procedure followe 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 e± ϋl. , 1983). The uterine tPA cDNA differed from melanoma tPA at several sites (predominantly in the 3' untranslated region of the clone) (from Reddy et 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, f lled 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 Mutants

Usefully positioned restriction enzyme recognition sites within the tPA cDNA were replaced with a synthetic oligonucleotide linker containing a different and unique restriction enzyme recognizition site (Xho I). A variety of linker lengths (8 mer, 10 mer and/or 12 mer) were introduced such that the reading frame of mutant protein product was maintained either as a simple linker-insert mutant or following deletion mutagenesis utilizing the original linker insertion mutants. As an example, a detailed method for the construction of the Bglll (nucleotide 115) Xho (8 mer) mutant will be given with reference to Figure 2.

lug 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 KP0 4 (pH 7.5), 6.6mM MgCl 2 , 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.

For those restriction enzymes generating 3' cohesive ends the ends are made blunt by utilizing the 3' to 5' exonuclease activity of T4 DNA polymerase as per the manufacturers instructions.

After incubating at room temperature for one hour the Klenow was heat inactivated at 65 β C for five minutes. To this mixture was added 100 pmoles of phosphorylated 8 mer Xho I linker (commercially available), ligation buffer (final concentration of 50mM Tris (pH 7.8), lOmM MgCl 2 , 20mM DTT, lmM ATP and 50μg ml "1 bovine serum albumin) and 200u of T. DNA ligase. The ligation was allowed to proceed overnight at 22°C.

The ligated DNA was phenol :chloroform:lAA 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 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 aliquot of the religated DNA was transfected into the

E___ 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 was analyzed by restriction enzyme analysis and the loss of the unique Bgl II site and its replacement by a unique Xho I site was confirmed.

Using this protocol Xho I linkers of various sizes (8 mer, 10 mer and/or 12 mer) were introduced into the following sites of the tPA cDNA.

Native Restriction Site Relative Location

Bgl II (115) - between the beginning of the mature processed tPA protein and the start of the finger domain

Sty I (527) - at the N-terminal beginning of the growth factor domain

BstX I (515) at the approximate center of the Kr ngle 1 domain

EcoRI (720 internal to the Kringle 2 domain

Sea I (879) at the C-terminal of the Kringle 2 domain

EcoRI (1201 internal to the protease domain

Sac I (1345) internal to the protease domain

Sty 1 (1652) internal to the protease domain

The numbers in parenthesis reflect the nucleotide position.

In the construction of the mutants encoding the heavy chain deletions the following plasmids were appropriately restriction enzyme digested and l gated as in the example given in Figure 2. For example in the generation of deletion mutant Bgl/Sty a plasmid containing an 8 mer Xhol linker at the Bg II (115) site was ligated with a plasmid containing an 8 mer Xhol linker at the Sty 1 (277) site via their now common Xhol cut cohesive ends.

Mutant Lesion Plasmids Utilized

Bgl/Sty del (5-57); 4 LEA 58 Bglll (115) Xhol (8 mer) Styl (277) Xhol (8 mer)

Bgl/Bstx del (5-139); 4 PLE 140 Bglll (115) Xhol (12 mer BstXI (515) Xhol (12 me

Bgl/Eco del (5-207); 4 PRG 208 Bglll (115) Xhol (10 me EcoRI (729) Xhol (10 me

Bgl/Sea del (5-258); 4 LED 259 Bglll (115) Xhol (8 mer) Seal (879) Xhol (8 mer)

Sty/Bstx del (58-139); 58 SPR 140 Styl (277) Xhol (10 mer) BstXI (515) Xhol (10 mer

Sty/Eco del (58-207); 57 SPRG 208 Styl (277) Xhol (10 mer) EcoRI (729) Xhol (10 mer

Sty/Sea del (58-258); 57 SLED 259 Styl (277) Xhol (8 mer) Seal (879) Xhol (8 mer)

Verification of mutants

All mutants were verified by restriction enzyme analysis, sequencing and/or in vitro transcription/translation analysis,

Mutant-encoded protei ns wi th suffi ci ent fi bri nolyti c activi ty were analyzed by zy ography rel ative to wi ld-type tPA.

Del etion mutaqenesi s of the heavy chai n of tPA

The utilization of appropriately positioned linker inserts (Xho I linkers) made it possible to generate a series of in-frame deletion mutants within the DNA encoding the heavy chain of tPA. An example of this deletion of the DNA encoding the "finger" domain of tPA is depicted in Figure 2 and shown on Table 3h). The Bgl II (115) and Sty I (277) sites are situated near to the boundaries of this domain. By the appropriate restriction enzyme digestion, gel electrophoresis and ligation, it was possible to generate a tPA cDNA which lacked the region of DNA encoding the finger domain (mutant Bgl/Sty on del(5-57); 4LEA58).

Bgl II ( 115) Xhol (8) Sty I (277 Xho I (8)

112 126 274 28

GCC . AGA . TCC . CTC . GAG . GGA . TCT . TAC . CAA- -GAG.CCA.AGC.CTC.GAG.GCA.AGG.TC al a arg ser l eu gl u gly ser tyr gi n— -glu pro ser leu glu ala arg cys

Xho I cut

28

TC.GAG.GCA.AGG.TG glu ala arg ay

Ligate

112 285

GCC.AGA.TCC.CTC.GAG.GCA.AGG.TGT ala arg ser leu glu ala arg cys

2 3 4 58 59

Bgl/Sty or del (5-57); 4 LEA 58

i.e. a deletion of amino acids 5 to 57 and an insertion of leu; glu and ala. (N.B. the finger domain includes amino acids 9 to 46).

The same procedure was used to generate the following heavy chain deletion mutants:

Domains Deleted

Table 3 shows the sequences of these and other mutants in detail. Table 4 provides Specific Activity data for the preferred embodiments of the present invention.

Verification of mutations

All mutants 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.

Vectors

Sp65-tPA

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 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 in v tro RNA synthesis and in 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

Mutated cDNA molecules were recloned into the LK444BHS vector as shown in Figure 3. The BamHI, Hindlll fragment of tPA cDNA or mutant derivative contained in the SP65 vector was 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

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 metal!othionine promoter in C127 cells.

CLH3AXSV2DHFR-tPA

The Sal I fragment containing the tPA cDNA or mutated derivative was isolated from the SP65 vector by restriction enzyme cleavage and gel purification. This fragment was ligated into Xho I cleaved vector ELH3AXSV2DHFR as shown in Figure 4. The orientation was determined and selected such that the inserted sequence was under the driving force of the mettallothionein promoter in CHO cells.

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 + 107« 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 mgDEAE-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 37°C for 4 hours. The cells v/ere 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 107. 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 KC1 , 0.05 g Na 2 HP0 4 , 0.5 g dextrose, and 2.5 g HEPES, pH 7.5 per 500 ml total volume). 30 μl of 2M CaCl 2 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 37 β C. The media was removed and the cells were then shocked with 15% glycerol in transfection buffer at 37 β C for 3.5 minutes. After 48 hours, the cells were split at a 1\3 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.

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. 4,656,134 to Ringold).

Transfection of C127 Cells

Mouse C127 cells were transfected with DNA prepartions according to methods previously published by researchers in

Assignees laboratories (Hsiung et al., 3. Mol. Appl. Genetics 2, 497 (1984)). Genes encoding modified tPA's were cloned into BPV-based vector CLH3AVBPV and these plasmids used for tranfections.

Modified tPA proteins were purified from conditioned medium by previously reported procedures (Lau et al., Bio/Technology 5, 953 (1987)).

Assays of Modified tPA's

Quant tation of mtPA proteins in conditioned medium was performed with a commercially available ELISA Kit for determination of tPA from American Diagnostica (Greenwich, CT, USA). The coating and detection antibody is a goat anti-human tPA IgG.

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 ;mearure 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

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 carried out for two currently used blood clot lysing 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, 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

For emergency treatment of thrombi by bolus injection, 5-1Omg of lyophilized mtPA are mixed together with saline and placed in th chamber of a syringe, which is used to inject the mtPA bolus into the patient intravenously.

Example 2

For infusion treatment for the rapid lysis of coronary thrombi, about lOOmg/hr of lyophilized mtPA are infused 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 in vitro assays and in. vivo imaging applications.

TABLE 1

TGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGT GGA i . + + + + + + 60

ACACTTCGTTAGTACCTACGTTACTTCTCTCCCGAGACGACACACGACGACGACACA CCT

START SIGNAL aa M K R G L C C V L L L C G - GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGATCT

61 . + + +— »+ +— . + 120

CGTCAG GCA GCGGGTCGGTCCT TAGGTACGGGCT GTCTTCTCCTCGGTCTAGA PROPEPTIDE <τl aa A V F V S P S Q E I H A R F R R G A R S -

TACCAAGTGΔTCTGCAGAGATGAAAAAACGCAGATGATATACCAGCAACATCAGTC ATGG

121 + + ._-—.+ — + + .. — + 180

ATGGTTCACTAGACGTCTCTACTTTTTTGCGTCTACTATATGGTCGTTGTAGTCAGT ACC r* aa Y Q V I C R D E K T Q M I Y Q Q H Q S W -

CTGCGCCCTGTGCTCAGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCAGG GCA

181 .— . + .--+ . -+— + + — + 240

GACGCGGGACACGAGTCTTCGTTGGCCCACCTTATAACGACCACGTTGTCACCGTCC CGT FINGER DOMAIN aa L R P V L R S N R V E Y C W C N S G R A -

CAGTGCCACTCAGTGCCTGTCAAAAGTTGGAGCGAGCCAAGGTGTTTCAACGGGGGC ACC 241 — + + ——-+ +- + + 300 GTCACGGTGAGTCACGGACAGTTTTCAACGTCGCTCGGTTCCACAAAGTTGCCCCCGTGG aa Q C H S V P V K S C S E P R C F N G G T

TGCCAGCAGGCCCTGTACTTCTCAGATTTCGTGTGCCAGTGCCCCGAAGGATTTGCT GGG 301 + + + + + + 360

ACGGTCGTCCGGGACATGAAGAGTCTAAAGCACACGGTCACGGGGCTTCCTAAACGA CCC

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

361 . + + . + + + + 420

TTCACGACACTTTATCTATGGTCCCGGTGCACGATGCTCCTGGTCCCGTAGTCGATG TCC < ~ & aa K C C E I D T R A T C Y E D Q G I S Y R -

GGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCG TTG 421 + + + + »-+ + 480

CCGTGCACCTCGTGTCGCCTCTCACCGCGGCTCACGTGGTTGACCTTGTCGTCGCGC AAC aa G T W S T A E S G A E C T N W N S S A L -

SUBSTITUTESHEET

GCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAACCAC

481 — + + + + + ~+ 540

CGGGTCTTCGGGATGTCGCCCGCCTCCGGTCTGCGGTAGTCCGACCCGGACCCCTTG GTG KRINGLE 1 DOMAIN aa A Q K P Y S G R R P D A I R L G L G N H -

AACTACTGCAGA CCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCGGGG

541 _ ___.___.__.+_ +_____-_ + __. +_ __+___—__. + 600 TTGATGACGTCTTTGGGTCTAGCTCTGAGTTTCGGGACCACGATGCAGAAATTCCGCCCC aa N Y C R N P D R D S K P W C Y V F K A G - GTACAGCTCAGAGTTCTGCAGCACCCCTGCCTGCTCTGAGGGA CAGTGACTGCTAC 601 + + + • — -+ + + 660

TTCATGTCGAGTCTC GACGTCGTGGGGACGGACGAGACTCCCT TGTCACTGACGATG aa K Y S S E F C S T P A C S E G N S D C Y - TTTGGG TGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGC

661 ■ + ■ + •+ — — + + — •+ 720

AAACCCTTACCCAGTCGGATGGCACCGTGCGTGTCGGAGTGGCTCAGCCCACGGAGG ACG aa F G N G S A Y R G T H S L T E S G A S C -

CTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGT GCC

721 + + + + + + 780

GAGGGCACCl^ GGTACTAGGACTATCCGTTCCAAATGTGTCGTGTCTTGGGGTCACGG KRINGLE 2 DOMAIN aa L P W N S M I L I G K V Y T A Q N P S A -

CAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAG CCC

781 + + + + + + 840

GTCCGTGACCCGGACCCGTTTGTATTAATGACGGCCTTAGGACTACCCCTACGGTTC GGG aa Q A L G L G K H N Y C R N P D G D A K P

TGGTGCCACGTGCTG G CCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGC

841 + + + + + + 900 ACCACGGTGCACGACTTCTTGGCGTCCGACTGCACCCTCATGACACTACACGGGAGGACG aa W C H V L K N R R L T tf E Y C D V P S C ϊ

TCCΔCCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCA GGAGGGCTCTTC 901 + + + + + + 960

AGGTGGACGCCGGACTCTGTCATGTCGGTCGGAGTCΔAAGCGTAGTTTCCTCCCGA GAAG aa S T C G L R Q Y S Q P Q F R I K G G L F

SUBSTITUTE SHEET

GCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCC

961 »-+ — + +- + + + 1020

CGGCTGTAGCGGAGGGTGGGGACCGTCCGACGGTAGAAACGGTTCGTGTCCTCCAGC GGG PROTEASE DOMAIN aa A D I A S H P W Q A A I F A K H R R S P -

GGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCC GCC

1021 ■■ — — +- + + + + + 1080 CCTCTCGCCAAGGACACGCCCCCGTATGAGTAGTCGAGGACGACCTAAGAGAGACGGCGG aa G E R F L C G G I L I S S C W I L S A A -

CACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACA TAC 1081 —— + ■ —+• + + + • + 1140

GTGACGAAGGTCCTCTCCAAAGGCGGGGTGGTGGACTGCCACTAGAACCCGTCTTGT ATG aa H C F Q E R F P P H H L T V I L G R T Y - CGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAG

1141 _ .+_„___.___+ + +_.. +_ + 1200

GCCCACCAGGGACCGCTCCTCCTCGTCrrTTAAACTTCAGCTTTTTATGTAACAGGT ATTC aa R V V P G E E E Q K F E V E K Y I V H K

GAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGAT TCG

1201 — +- . >--+ + + + + 1260

CTTAAGCTACTACTGTGAATGCTGTTACTGTAACGCGACGACGTCGACTTTAGCCTA AGC aa E F D D D T Y D N D I A L L Q L K S D S -

TCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGAC CTG

1261 + + • + + + + 1320

AGGGCGACACGGGTCCTCTCGTCGCACCAGGCGTGACACACGGAAGGGGGCCGCCTG GAC aa S R C A Q E S S V V R T V C L P P A D L t

CAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTG TCT

1321 + -+ + + + + 1380 GTCGACGGCCTGACCTGCCTCACACTCGAGAGGCCGATGCCGTTCGTACTCCGGAACAGA aa Q L P D W T E C E L S G Y G K H E A L S

CCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGC TGC 1381 + + ■ + + + + 1440

GGAAAGATAAGCCTCGCCGACTTCCTCCGAGTACAGTCTGACATGGGTAGGTCGGCG ACG aa P F Y S E R L K E A H V R L Y P S S R C -

SUBSTITUTE SHEET

ACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACT 1441 + + + + + + 1500

TGTAGTGTTGTA TG TTGTCTTGTCAGTGGCTGTTGTACGACACACGACCTCTGTGA aa T S Q H L L N R T V T D N M L C A G D T -

CGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGC CCC 1501 - + + _+____ .+__,„___,„__+_____,__._____+ 1560 GCCTCGCCGCCCGGGGTCCGTTTGAACGTGCTGCGGACGGTCCCGCTAAGCCCTCCGGGG aa R S G G P Q A N L H D A C Q G D S G G P -

CTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTG GGC 1561 + + + + + + 1620

GΔCCACACAGACTTGCTACCGGCGTACTGAAACCACCCGTAGTAGTCGACCCCGGA CCCG aa L V C L N D G R M T L V G I I S W G L G TGTGGACAGAAGGATGTCCCGGGTGTGTACACCAAGGTTACCAACTACCTAGACTGGATT

1621 + • + »+ + .-——+. + 1680

ACACCTGTCTTCCTACAGGGCCCACACATGTGGTTCCAATGGTTGATGGATCTGACC TAA aa C G Q K D V P G V Y T K V T N Y L D W I

CGTGACAACATGCGACCGTGACCAGGAACACCCGACTCCTCAAAAGCAAATGAGATC CCG

1681 + + + + + +

GCΔCTGTTGTACGCTGGCACTGGTCCTTGTGGGCTGAGGAGTTTTCGTTTACTCTA GGGC

«l aa R D N M R P END

CCTCTTCTTCOTCAG GACACTGCA GGCG^^ 1741 + + + +— + + 1800

GGAG GAAG GTCTTCTGTGACGTTTCCGCGTCACGAAGAGATGTCTGAAGAGGTCTG

CCACCACACCGCAGAAGCGGGACGAGACCCTACAGGAGAGGGAAGAGTGGCATTTTC CCA 1801 + ■+- + + + + 1860

GGTGGTGTGGCGTCI CGCCCTGCTCTGGGATGTCCTCTCCCTTCTCACCGTAAAAGGGT GATACTTCCCATTTTGG GATTTCAGGACTTGGTCTGATTTCAGGATACTCTGTCAGAT

1861 + + + + + + 1920

CTATGAAGGGTAA CCTTCTAAAGTCCTGAACCAGACTAAAGTCCTATGAGACAGTCTA

GGGAAGACATGAATGCACACTAGCCTCTCCAGGAATGCCTCCTCCCTGGGCAGAAAT GGC 1921 + + + + . -+ + 1980

CCCTTCTGTACTTACGTGTGATCGGAGAGGTCCTTACGGAGGAGGGACCCGTCTTTA CCG

CATGCCACCCTGTTTTCAGCTΔAAGCCCAACCTCCTGACCTGTCACCGTGAGCAGC TTTG 1981 + + +- + + - .— + 2040 GTACGGTGGGACAAAAGTCGATTTCGGGTTGGAGGACTGGACAGTGGCACTCGTCGAAAC

SUBSTITUTE SHEET

GAAACAGGACCACAAAAATGAAAGCATGTCTCAATAGTAAAAGATAACAAGATCTTTCAG 2041 —■ + + ■ + + + + 2100

CTTTGTCCTGGTGTTTTTACTTTCGTACAGAGTTATCATTTTCTATTGTTCTAGAAA GTC

GAAAGACGGATTGCATTAGAAATAGACAGTATATTTATAGTCACAAGAGCCCAGCAG CGG 2101 —— + + + __+__. + __.+ 2160

CTTTCTGCCTAACGTAATCTTTATCTGTCATATAAATATCAGTGTTCTCGGGTCGTC GCC CTCAAAGTTGGGGCAGGCTGGCTGGCCCGTCATGTTCCTCAAAAGAGCCCTTGACGTCAA

2161 » +■— +—« + » + + + 2220

GAGTTTCAACCCCGTCCGACCGACCGGGCAGTACAAGGAGTTTTCTCGGGAACTGCA GTT

GTCTCCTTCCCCTTTCCCCACTCCCTGGCTCTCAGAAGGTATTCCTTTTGTGTACAG TGT 2221 _ +.—. + _+__.-__._____+ + + 2280

CAGAGGAAGGGGAAAGGGGTGAGGGACCGAGAGTCTTCCATAAGGAAAACACATGTC ACA

GTAAAGTGTAAATCCTTTTTCTTTATAAACTTTAGAGTAGCATCGAGAGAATTGTAT CAT 2281 —— + + + + + + 2340 CAΓΓTCACATTTAGGAAAAAGAAATATTTGAAATCTCATCGTAGCITCTCTTAACATA GTA

TTGAACAACTAGGCTTCAGCAΔTATTTATAGCAATCCATAGTTAGTTTTTACTTTT CGTT

2341 + + .. + + + + 2400

AACTTGTTGATCCGAAGTCGTTATAAATATCGTTAGGTATCAATCAAAAATGAAAAG CAA

GCCACAACCCTGTTTTATACTGTACTTAATAAATTCAGATATATTTTTCACAGTTTT TCC

2401 +_—. +—.—.___._.+___._ + + + 2460

CGGTGTTGGGACAAAATATGACATGAATTATTTAAGTCTATATAAAAAGTGTCAAAA AGG

SUBSTITUTE SHEET

TABLE 2

DESIGNATION OF TPA DOMAINS (after Degan et al. 1986)

TABLE 3

(All non-primed letters (e.g., a)) refer to nucleotide sequences, all primed letters (e.g., a )) refer to corresponding amino acid sequences)

Bglll (115) Xho I (8 mer) 4LEGS5

103 126 a ) AGA.AGA.GGA. GCC .AGA.TCC.CTC.GAG.GGA.TCT.TAC.CAA a ) arg arg gly ala arg ser leu αlu αlv ser tyr gin, -2 -1 1 2 3 4 5 6

StvI(277)Xho I (8 mer) 57SLEA58

268 288 b ) TGC.AGC.GAG.CCA.AGC.CTC.GAG.GCA.AGG.TGT.TTC b 1 ) cys ser glu pro ser leu αlu ala arg cys phe 54 55 56 57 58 59 60

BstXI(515)Xho I (10 mer) del(138-139) 137 PR140

505 540 c ) AGG.CCA.GAC.GCC.ATC.CCT.CGA.GGG.CTG.GGG.AAC.CAC c ) arg pro asp ala ile pro arg gly leu gly asn his 133134135136137 140141 142143144

EcoRI(1201)Xho I (8 mer) 365 FLEE 366

1192 1215 d ) GTC.CAT.AAG.GAA.TTC.CTC.GAG.GAA.TTC.GAT.GAT.GAC d, l ) val his lys glu phe leu glu glu phe asp asp asp

362 363 364365 366367 368369

SacI(1345)Xho I (10 mer) del(413-414): 412 ALEG415

1330 1359 e ) GAC . TGG . ACG . GAG . TGT . GCC. CTC. GAG. GGC . TCC . GGC . TAC e 1 ) asp trp thr glu cys ala l eu glu gl v ser gly tyr 408 409 410 411 412 415 416 417

Styl(1652) Xho I (10 mer) 516 PRGK 517

1639 1665 f ) CCG.GGT.GTG.TAC.ACC.AAG.CCT.CGA.GGC.AAG.GTT.ACC.AAC

j f ) pro gly val tyr thr lys pro arg gl v Ivs val thr asn

511 512 513 514 515 516 517 518 519

Sea I(879)Xho I (12 mer) del 258: 257 SPRGD 259

868 891 g ) CTG.ACG.TGG.GAG.TCC.CCT.CGA.GG .GAC.TGT.GAT.GTG g ) leu thr trp glu ser pro arα αlv asp lys asp val

254255256257 259260261

Bglv/Stv del(5-57):4LEA58

106 120 280 288 h ) AGA.GGA.GCC.AGA.TCC.CTC.GAG.GCA.AGG.TGT.TTC h ) arg gly ala arg ser leu glu ala arg cys phe

-1 1 2 3 4 58 59 60

Bgl/Bstx deK5-139):4PLE140 106 120 539 543 i ) AGA.GGA.GCC.AGA.TCC.CCC.CTC.GAG.GGG.CTG.GGG.AAC.CAC.AAC

j i ) arg gly ala arg ser pro leu αlv gly leu gly asn his asn

-1 1 2 3 4 140 141 142 143 144 145

Bαl/Eco del(5-207):4PRG208

106 120 730 741 j ) AGA.GGA.GC .AGA.TC . CT.GCA.GGG.AAT.TC .ATG.ATC

j j ) arg gly ala arg ser pro arg gly asn ser met ile

-1 1 3 4 208209 210211

Bαl/Sca del(5-258):4LED259

106 120 883 894 k ) AGA.GGA.GCC.AGA.TCC.CTC.GAG.GAC.TGT.GAT.GTG. CC k ) arg gly ala arg ser leu glu asp cys asp val pro

-1 1 2 3 4 259260261 262

Stv/Bstx del(58-139):58 SPR140

268 281 539 553

1 ) TGC.AGC.GAG.CCA.AGC.CCT.CGA.GGG.CTG.GGG.AAC.CAC.AAC

1 ) cys ser glu pro ser pro arg gly leu gly asn his asn

54 55 56 57 140 141 142 143 144 145

Stv/Eco del(58-207):57SPRG208

268 279 730 741 m ) TGC.AGC.GAG.CCA.AGC.CCT.CG .GGG.AAT.TCC.ATG.ATC m 1 ) cys ser glu pro ser pro arg glv asn ser met ile

54 55 56 57 208209210211

Sty/Sea del(58-258):57SLED259 268 279 883 891 n ) TGC.AGC.GAG.CCA.AGC.CTC.GAG.GAC.TGT.GAT.GTG n 1 ) cys ser glu pro ser leu glu asp cys asp val

54 55 56 57 259260261

(mer specifications in paranthesis indicate length of necessary linker which, when inserted at the specified site, results in correct reading frame for translation.)

TABLE 4

Mutant Speci fi c Acti vi ty.?

W ld type 693 Bglll (115) 657 Styl (277) 247

10 BstXI (515) 217 EcoRI (729) 159 Seal (879) 237 EeoRI (1201) 76 Sad (1345) 8

15 Styl (1652) NPD Bgl/Sty 126 Bgl/BstX 141 Bgl/RI 195 Bgl/Sea 13

20 Sty/RI NPD* Sty/Sea NPD*

# - IU/μg in indirect amidolytic assay

25

NPD - Not Possible to Determine

*These molecules demonstrated low fibrinolysis activity 125 by I fibrin lysis assay

30