WO1988003171A1

WO1988003171A1 - Human pancreatic secretory trypsin inhibitors produced by recombinant dna methods and processes for the production of same

Info

Publication number: WO1988003171A1
Application number: PCT/US1987/002585
Authority: WO
Inventors: Tadahiko Kohno; Pradip Bandyopadhyay; Stephen P. Eisenberg; Robert C. Thompson
Original assignee: Synergen Biologicals, Inc.
Priority date: 1986-10-30
Filing date: 1987-10-09
Publication date: 1988-05-05
Also published as: EP0329693A4; AU8172787A; KR880701777A; EP0329693A1; JPH02501027A

Abstract

A protein possessing at least one active site capable of inhibiting trypsin and preventing trypsin activation of zymogen precursors of hydrolytic enzymes, along with recombinant methods for the production of this enzyme. Analogs of this inhibitor which also possess the ability to inhibit trypsin are also disclosed. Certain of these analogs additionally possess an enhanced ability to inhibit various proteolytic enzymes, such as elastase.

Description

HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITORS PRODUCED BY RECOMBINANT DNA METHODS AND PROCESSES FOR THE PRODUCTION OF SAME BACKGROUND OF THE INVENTION The present invention relates to a recombinant DNA method for the production of human pancreatic secretory trypsin inhibitors (HPSTIs) and analogs thereof and methods for the production of HPSTI and its analogs using microbial recombinant DNA expression systems.

The pancreas is an organ which secretes various types of enzymes, including amylolytic, lipolytic and proteolytic enzymes. The proteolytic enzymes secreted by the pancreas include endopeptidases such as trypsin, chymotrypsin, elastase and several exopeptidases. Since these proteolytic enzymes are capable of digesting peptide bonds and are therefore capable of digesting the pancrease itself, they are produced as inactive precursors known as zymogens. The transformation of these zymogens into the active proteolytic enzyme is prevented by the synthesis of the proteolytic enzymes in zymogen forms, by their packaging in zymogen granules, and by the presence of protease inhibitors, particularly trypsin inhibitors, which prevent small amounts of trypsin from activating the zymogen.

Certain disease states of the pancreas allow activation of the zymogen forms of the proteolytic enzymes which results in autodigestion of the pancreas and associated inflammation and disorders of the circulatory system. These disease states may be caused by a variety of factors, including acute or chronic alcohol abuse, biliary tract disease, postoperative complications or hyperlipidemia.

In an effort to arrest the autodigestion of the pancreas caused by these disease states, methods of treatment have been sought which would be capable of halting uncontrolled activation of the pancreatic proteolytic enzymes. In this regard, attempts have been made to use bovine pancreatic trypsin inhibitor intravenously to control the activation of the proteolytic enzymes. However, the treatment has not been clearly effective and the human immune system has occasionally recognized this protein as foreign and has mounted an immune response to the very substance which is being used as a treatment agent. To overcome the problem created by the adverse immune reaction arising from the use of forexgn proteins, it has been postulated that a human trypsin inhibitor, in particular HPSTI, be investigated as a treatment agent. In this regard, HSPTI has been identified and its amino acid sequence has been described by Bartelt, D.C., et al. in "The Primary Structure of the Human Pancreatic Secretory Trypsin Inhibitor," Arch. Biochem. Biophys. 179; 189-199 (1977) and by Yamamoto, T., et al. in "Molecular Cloning and Nucleotide Sequence of Human Pancreatic Secretory Inhibitor (PSTI) cDNA," Biochem. and Biophys. Res. Comm. 132:605-612 (1985). However, in spite of these descriptions of HPSTI, to date HPSTI has been isolatable only from human pancreatic juices and tissue. Thus, HPSTI has not been available in quantities sufficient for use in testing to determine its efficacy as a treatment agent.

Surprisingly, the present inventors have discovered a DNA sequence encoding HPSTI and, using this sequence, recombinant DNA methods for producing HPSTI in microbial expression systems. The HPSTI thus produced is biologically equivalent to that isolated from human pancreatic juices and tissues. Additionally, the present inventors have discovered analogs of HPSTI which also are believed to be useful in treating and preventing autodigestion of the pancreas and for treatment of other diseases known to involve the uncontrolled destruction of tissue by proteases.

The HPSTI and the analogs thereof of the present invention, prepared by the recombinant DNA methods set forth herein, will not only enable improved research into the prevention and treatment of diseases resulting in the autodigestion of pancreatic tissue but will serve as an efficacious replacement for the current treatment methods of dubious efficacy utilizing bovine pancreatic trypsin inhibitor. Therefore, it is believed that future treatment methods for diseases involving autodigestion of pancreatic tissue will include the use of HPSTI or an analog thereof to prevent the trypsin-catalyzed activation of pancreatic proteolytic zymogens.

SUMMARY OF THE INVENTION

Present invention relates to human pancreatic secretory trypsin inhibitor (HPSTI) and analogs thereof, recombinant DNA methods for producing the same and to portable DNA sequences capable of directing microbial production of HPSTI and its analogs. The present invention also relates to a series of vectors containing these portable DNA sequences.

One object of the present invention is to provide a human pancreatic secretory trypsin inhibitor, produced by recombinant DNA methods, which can be produced in sufficient quant i t i es and purities to allow the production of pharmaceutical compositions which are useful in the protection of pancreatic tissues from autodigestion by native, activated pancreatic enzymes.

An additional object of the present invention is to provide a recombinant DNA method for the production of HPSTI. The recombinant HPSTIs produced by this method are intended to be biologically equivalent to HPSTI isolatable from human pancreatic juices and tissues.

Another object of the present inven-tion is to provide analogs of HPSTI and recombinant DNA methods for the production of these analogs. Some of these HPSTI analogs are capable of inhibiting trypsin, thus preventing activation of pancreatic proteolytic zymogens and the consequent autodigestion of the pancreas. Others are capable of inhibiting various proteolytic enzymes which are involved in destruction of other tissues.

To facilitate the recombinant DNA synthesis of HPSTI and analogs thereof, it is a further object of the present invention to provide portable DNA sequences capable of directing production of these proteins. Moreover, it is also an object of the present invention to provide cloning vectors containing these portable sequences. These cloning vectors are capable of being used in microbial expression systems to produce pharmaceutically useful quantities of HPSTI and its analogs.

A further object of the present invention is to provide pharmaceutically-useful preparations containing, as at least one of the active ingredients, the HPSTI or HPSTI analogs of the present invention.

Additional objects and advantages of the present invention will be set forth in part in the description which follows, or may be learned from practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalitxes and combinations particularly pointed out in the appended claims.

To achieve the objects and in accordance with the purposes of the present invention, HPSTIs produced by recombinant DNA methods are set forth. This HPSTI is remarkably similar to the natural HPSTI isolatable from pancreatic tissues and juxces. Moreover, this HPSTI is biologically equivalent to that isolated from human pancreatic juices and tissues.

Analogs of HPSTI are also set forth. Some of these analogs are biologically equivalent to HPSTI isolatable from human pancreatic tissues and juices in their ability to inhibit trypsin. Other analogs are capable of inhibiting proteolytic enzymes other than trypsin to prevent destruction of various tissues. These enyzmes include, for example, elastase which has been implicated in a causative role in emphysema. Recombinant DNA methods for producing these HPSTI analogs are also disclosed.

To further achieve the objects and in accordance with the purposes of the present invention, as embodied and broadly described herein, portable DNA sequences encoding HPSTI and the disclosed analogs are provided. These portable DNA sequences comprise nucleotide sequences capable of directing production of either HPSTI or its analogs. The portable DNA sequences may be either synthetically-produced sequences or restriction fragments (hereinafter referred to as "natural" DNA sequences). The synthetic DNA sequences may be prepared by polynucleotide synthesis sequencing and techniques known to those of ordinary skill in the art.

In a preferred embodiment, a portable DNA sequence encoding HPSTI is isolated from a human pancreatic cDNA library and is capable of directing production of HPSTI which is biologically equivalent to that which is isolatable from human pancreatic juices and tissues. In the synthetic embodiment, the coding strand of a first preferred DNA sequence which has been discovered has the following nucleotide sequence: GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC GGT TGC ACT AAA ATC TAC AAC CCG GTA TGT GGT ACC GAC GGT GAC ACC TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT CAG ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

A second preferred DNA sequence has also been discovered which has the following nucleotide sequence: GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC GGT TGC ACT AAA ATC TAC GAC CCG GTC TGC GGT ACC GAT GGT AAC ACC TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT CAG ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

The nucleotides represented by the foregoing abbreviations are set forth below in the Detailed Description of the Preferred Embodiment.

Additionally, portable DNA sequences useful in the processes of the present invention to create HPSTI analogs are set forth. A preferred portable DNA sequence of this embodiment of the present invention is created by site directed mutagenesis of the sequence set forth above and has the lysine in the 18th position changed to either an arginine, methionine, valine, leucine alanine, phenylalanine, tryptophan or tyrosine. Furthermore, to achieve the objects and in accordance with the purposes of the present invention, recombinant DNA methods are disclosed which result in the manufacture, from microbial cells, of the instant HPSTI or HPSTI analogs using the portable DNA sequences referred to above. In one embodiment, this recombinant DNA method comprises:

(a) preparation of a portable DNA sequence capable of directing a host microorganism to produce a human pancreatic secretory trypsin inhibitor or analog thereof;

(b) cloning the portable DNA sequence into a vector capable of being transferred into and replicating in a host microorganism, such vector containing operational elements for the portable DNA;

(c) transferring the vector containing the portable DNA sequence and operational elements into a host microorganism capable of expressing the human pancreatic secretory trypsin inhibitor gene;

(d) culturing the host microorganism under conditions appropriate for amplification of the vector and expression of the inhibitor; and

(e) harvesting the inhibitor in an active form. Moreover, the present invention provides a method for producing a pharmaceutically-acceptable composition using this recombinant-DNA method and further combining the resultant inhibitor with one or more pharmaceutically-acceptable carriers.

To further accomplish the objects and in further accord with the purposes of the present invention, a serxes of expression vectors is provided comprising at least one of the portable DNA sequences discussed above. In particular, vectors pSGE1 and pSGE9 are disclosed.

It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate two embodiments of the invention, and, together with the description, serve to explain the principles of the invention.

A BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a restriction map of plasmid pSGE1.

Figure 2 is a restriction map of plasmid pSGE9.

Figure 3 is a growth curve of SGE45. DETAILED DESCRIPTION OF THE PREEERRED EMBODIMENT

Reference will not be made in detail to the presently preferred embodiments of the invention, which, together with the drawings and the following examples, serve to explain the principles of the invention.

As noted above, the present invention relates in part to portable DNA sequences capable of directing microbial production of human pancreatic secretory trypsin inhibitors (HPSTIs) and analogs thereof in a variety of microbial systems. "Portable DNA sequence" in this context is intended to refer either to a synthetically-produced nucleotide sequence or to a suitably modified restriction fragment of a naturally-occurring DNA sequence.

For purposes of this specification, the term "human pancreatic secretory trypsin inhibitor" or "HPSTI" is intended to mean the protein whose primary structure was established by Bartelt et al., supra., by Yamamoto et al., supra, or a protein which is defined by the codons present in a deoxyribonucleic acid sequence, including the sequences listed above, which proteins function in a manner which is biologically equivalent to native HPSTI, and which may or may not include post-translational modifications and any other similar protein with the same function that may be discovered in the human pancreas. It is further intended that the term "human pancreatic secretory trypsin inhibitor" or "HPSTI" refers to either the form of the protein that would be excreted from a microorganism or the methionyl-inhibitor as it may be present in microorganisms from which it was not excreted.

Moreover, it must be borne in mind that the present invention encompasses pharmaceutically-acceptable compositions containing, as at least one of the active ingredients, the HPSTI or HPSTI analogs of the present invention. In this embodiment, the compositions would also contain one or more compounds selected from the group consisting of physiological saline, fillers or binders such as starch, disintegrators and flavoring agents. It is contemplated that these pharmaceutically-acceptable compositions may be in the form of tablets, capsules, syrups, or solutions suitable for intramuscular, intraperitoneal or intravenous injection.

In the practice of this invention, alteration of some amino acids in a protein sequence may occur so long as the alterations do not affect the fundamental properties of the claimed proteins. Therefore, it is also contemplated that other portable DNA sequences, both those capable of directing production of identical amino acid sequences and those capable of directing production of analogous amino sequences which also possess the desired activity, are included within the ambit of the present invention.

It is contemplated that some of these analogous amino acid sequences will be substantially homologous to the native HPSTI while other amino acid sequences, capable of functioning as a trypsin inhibitor, will not exhibit substantial homology to native HPSTI. By "substantial homology," as used herein, is meant a degree of homology to a native HPSTI in excess of 75%, preferrably in excess of 80%, and more preferrably in excess of 90%. The percentage homology as discussed herein is calculated as a percentage of the amino acid residues found in the smaller of the two sequences that align with identical amino acid residues in the sequence being compared when two gaps in a length of 50 amino acids may be introduced to assist in that alignment as set forth by Dayhoff, M.O., in Atlas of Protein Sequence in Structure, Volume 5, page 124 (1972), National Biochemical Research Foundation, Washington, D.C., specifically incorporated herein by reference.

I. Portable DNA Sequences

In a preferred embodiment, the portable DNA sequences are capable of directing microbial production of HPSTI. These HPSTIs are biologically equivalent to that previously isolated from human pancreatic juices and tissues. By "biologically equivalent," as the term is used herein in the specification and the claims, it is meant that an inhibitor, produced using a portable DNA sequence of the present invention, is capable of preventing trypsin activation of the zymogen precursors of proteolytic enzymes of the same type, but not necessarily to the same degree, as a native human pancreatic secretory trypsin inhibitor , specifically that HPSTI isolatable from human pancreatic juices and tissues.

A first portable DNA sequence of the present invention capable of directing production of HPSTI has a nucleotide sequence as follows:

GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC GGT TGC ACT AAA ATC TAC AAC CCG GTA TGT GGT ACC GAC GGT GAC ACC TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT CAG ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC wherein the nucleotides are represented by the abbreviations indicated below.

NUCLEOTIDES ABBREVIATION Deoxyadenylic acid A

Deoxyguanidylic acid G

Deoxycytidylic acid C

Deoxythymidylic acid T

A second portable DNA of the present invention capable of directxng productxon of HPSTI, has a nucleotide sequence as follows:

GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC GGT TGC ACT AAA ATC TAC GAC CCG GTC TGC GGT ACC GAT GGT AAC ACC TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT CAG ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

As noted above, the portable DNA sequences of the present invention may be isolated from naturally-occurring DNA sources of may be synthetically created. It is believed that the means for synthetic creation of these polynucleotide sequences are generally known to one of ordinary skill in the art, particularly in light of the teachings contained herein. As an example of the current state of the art relating to polynucleotide synthesis, one is directed to Matteucci, M.D., and Caruthers, M.H., in J. Am. Chem. Soc. 103:3185 (1981) and Beaucage, S.L. and Caruthers, M.H., in Tetrahedron Lett. 22:1859 (1981), specifically incorporated herein by reference. The present invention also relates to portable DNA sequences capable of directing microbial production of the HPSTI "analogs" disclosed herein. As noted above, some of these analogs xnclude amino acid sequence wherexn the lysine in the 18th position is replaced with an arginine, methionine, valine, leucxne, alanine, phenylalanine, tryptophan or tyrosine. These substitutions preferably are accomplished by substitution, for the lysine codon at the 52nd through 54th nucleotides in the preferred sequence set forth above, of nucleotides encoding either arginine, methionine, valine, leucine, alanine, phenylalanine, tryptophan or tyrosine. The nucleotides suitable for these substitutions are known to those of ordinary skill in the art, particularly in view of references such as Grantham, R. et al. Nucleic Acids Research 9:r43 (1981).

It should also be noted that, due to codon degeneracy, other codons encoding the same amino acids may be substituted to obtain the same analogs. Such codons substitutions are readily known to those of ordinary skill in the art. II. The Vectors and Host Organisms

The present invention also relates to a series of vectors, each containing at least one of the portable DNA sequences described herein. It is contemplated that additional copies of the portable DNA sequence may be included in a single vector to increase a host microorganism's ability to produce large quantities of the desired HPSTI or the analogs thereof.

In addition to the portable DNA sequence, the cloning vectors within the scope of present invention may contain supplemental nucleotide sequences preceding or subsequent to the portable DNA sequence. These supplemental sequences are those which will not interfere with transcription of the portable DNA sequence and will, in some instances as set forth more fully below, enhance transcription, translation, secretion or the ability of the polypeptide chain of the resultant inhibitor to assume an active, tertiary structure.

A. Vector pSGEl

A preferred vector of the present invention is set forth in Figure 1. This vector, pSGEl, contains the preferred portable DNA sequence set forth above as encoding HPSTI.

The plasmid pSGEl is a derivative of pUC8 in which the DNA between the EcoRI and Sall sites in the polylinkef has been replaced with an EcoRI-Sall fragment containing the gene for bla-HPSTI. Amp^r as indicated in Fig. 1 is the betalactamase gene; P_LAC indicates a DNA segment containing the lac promoter, lac operator and coding sequences for the first six amino acids of beta galactosidase; bla-HPSTI indicates a DNA segment containing the beta Iactamase ribosome binding site and coding sequences for a beta Iactamase leader peptide/HPSTI fusion protein; B-gal indicates a DNA sequence containing sequences coding for the alpha complementing peptide of B-galactosidase; RI and Sall indicate recognition sites for restriction enzymes EcoRI and Sall; ORI indicates the Col E1 origin of replication; and the arrows show directions of transcription. B. Vector pSGE9

A second preferred vector, pSGE9, is set forth in Figure 2. It was constructed by replacing the DNA in the polylinker of pKK223-3 between the EcoRI site and the PstI site with an EcoRI-PstI fragment containing the gene for OmpA-HPSTI. In Figure 2, Amp^r is the beta Iactamase gene; P_tac contains DNA for the tac promoter, lac operator, and beta-galactosidase Shine/Dalgarno sequence; Ompa-HPSTI indicates a DNA segment containing 52 base pairs of DNA flanking the translational initiation codon upstream of the gene (and including the OmpA Shine/Dalgarno sequence) and coding sequences for an OmpA leader peptide/HPSTI fusion protein. RI, Pst, and Bam are recognition sites for EcoRI, PStI, and BamHI. Tet^r is a part of the gene from pBR322 which confers tetracycline resistance (therefore, this vector does not confer resistance to tetracycline); rrnB contains the DNA from the E . coli rrnB operon from position 6416 to 6840 including the transcriptional terminator. Ori indicates the ColEI origin of replication. Arrows indicate the direction of transcription.

C. Characteristics of Preferred Vectors Certain embodiments of the present invention are envisioned which employ known or currently undiscovered vectors which would contain one or more of the portable DNA sequences described herein. In particular, it is preferred that these vectors have some or all of the following characteristics: (1) possess a minimal number of host-organism sequences; (2) be stably maintained and propagated in the desired host; (3) be capable of being present in a high copy number in the desired host; (4) possess a regulatable promoter positioned so as to promote transcription of the gene of interest; (5) have at least one DNA sequence coding for a selectable trait present on a portion of the plasmid separate from that where the portable DNA sequence will be inserted; and (6) a DNA sequence capable of terminating transcription.

In various preferred embodiments, these cloning vectors containing and capable of expressing the portable DNA sequence of the present invention contain various operational elements. These "operational elements," as discussed herein, include at least one promoter, at least one Shine-Dalgarno sequence and initiator codon, and at least one terminator codon. Preferably, these "operational elements" also include at least one operator, at least one leader sequence for proteins to be exported from intracellular space, at least one gene for a regulator protein, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector DNA.

Certain of these operational elements may be present in each of the preferred vectors of the present invention. It is contemplated any additional operational elements which may be required may be added to these vectors using methods known to those of ordinary skill in the art, particularly in light of the teachings herein.

In practice, it is possible to construct each of these vectors in a way that allows them to be easily isolated, assembled, and interchanged. This facilitates assembly of numerous functional genes from combinations of these elements and the coding region of the HPSTI or HPSTI analog. Further, many of these elements will be applicable in more than one host. It is additionally contemplated that the vectors, in certain preferred embodiments, will contain DNA sequences capable of functioning as regulators ("operators"), and other DNA sequences capable of coding for regulator proteins.

1. Regulators

These regulators, in one embodiment, will serve to prevent expression of the portable DNA sequence- in the presence of certain environmental conditions and, in the presence of other environmental conditions, will allow transcription and subsequent expression of the protein coded for by the portable DNA sequence. In particular, it is preferred that regulatory segments be inserted into the vector such that expression of the protable DNA sequence will not occur, or will occur to a greatly reduced extent, in the absence of, for example, isopropylthio- -D-galactoside. In this situation, the transformed microorganisms containing the portable DNA may be grown to a desired density prior to initiation of the expression of the HPSTI or HPSTI analog. In this embodiment, expression of the desired trypsin inhibitor is induced by addition of a substance to the microbial environment capable of causing expression of the DNA sequence after the desired density has been achieved.

2. Promoters

The expression vectors must contain promoters which can be used by the host organism for expression of its own proteins. While the lactose promoter systemn is used commonly, other microbial promoters have been isolated and characterized, enabling one skilled in the art to use them for expression of the HPSTI gene.

3. Transcription Terminator

The transcription terminators contemplated herein serve to stabilize the vector. In particular, those sequences as described by Rosenberg, M. and Court, D., in Ann. Rev. Genet. 13:319-353 (1979), specifically incorporated herein by reference, are contemplated for use in the present invention.

4. Non-Translated Sequence

It is noted that, in the preferred embodiment, it may also be desirable to reconstruct the 3' or 5' end of the coding region to allow incorporation of 3' or 5' non-translated sequences into the gene transcript. Included among these non-translated sequences are those which stabilize the mRNA [or enhance its translation] as they are identified by Schmeissner, U., McKenney, K., Rosenberg, M. and Court, D. in J. Mol. Biol. 176:39-53 (1984), specifically incorporated herein by reference.

5. Ribosome Binding Sites

The microbial expression of foreign proteins require certain operational elements which include, but are not limited to, ribosome binding sites. That particular element is a sequence which a ribosome recognizes and binds to in the initiation of protein synthesis as set forth in Gold, L., et al., Ann. Rev. Microbiol. 35:557-580 (1983); Marquis, D.M., et al., Gene 42: 175-183 (1986), specifically incorporated herein by reference.

The operational elements as discussed herein can be routinely selected by those of ordinary skill in the art in light of prior literature and the teachings contained herein. General examples of these operational elements are set forth in B. Lewin, Genes, Wiley & Sons, New York (1983), which is specifically incorporated herein by reference. Various examples of suitable operational elements may be found on the vectors discussed above and may be elucidated through review of the publications discussing the basic characteristics of the aforementioned vectors.

6 . Leader Sequence and Translational Coupler Additionally, it is preferred that DNA coding for an appropriate secretory leader (signal) sequence be present at the 5' end of the portable DNA sequence. The DNA for the leader sequence must be in a position which allows the production of a fusion protein in which the leader sequence is immediately adjacent to and covalently joined to the inhibitor, i.e., there must be no transcription or translation termination signals between the two DNA coding sequences. The presence of the leader sequence is desired in part for one or more of the following reasons. First, the presence of the leader sequence may facilitate host processing of the initial product to the mature HPSTI or HPSTI analog. In particular, the leader sequence may direct cleavage of the initial translation product by a leader peptidase to remove the leader sequence and leave a polypeptide with the amino acid sequence which has the potential of HPSTI activity. Second, the presence of the leader sequence may facilitate purification of the HPSTI or HPSTI analog, through directing the HPSTI or HPSTI analog out of the cell cytoplasm. In some species of host microorganisms, the presence of the appropriate leader sequence will allow transport of the completed protein into the periplasmic space, as in the case of some E. coli. In the case of certain E. coli, saccharomyces and strains of Bacillus and Pseudomonas, the appropriate leader sequence will allow transport of the protein through the cell membrane and into the extracellular medium. In this situation, the protein may be purified from extracellular protein. Thirdly, in the case of some of the HPSTI or HPSTI analogs prepared by the present invention, the presence of the leader sequence may be necessary to locate the completed protein in an environment where it may fold to assume its active structure, which structure possesses the appropriate inhibitory activity.

7. Translation Terminator

The translation terminators contemplated herein serve to stop the translation of mRNA. They may be either natural, Kohli, J., Mol. Gen. Genet. 182:430-439 (1981), or synthesized, Pettersson, R.F., Gene 24:15-27 (1983), both of which references are specifically incorporated herein by reference.

8. Selectable Marker

Additionally, it is preferred that the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host microorganism. In a particularly preferred embodiment of the present invention, the gene for ampicillin resistance is included in vectors pSGEl and pSGE9. In other plasmids, the gene for tetracycline resistance or the gene for chloramphenicol resistance is included.

Such a drug resistance or other selectable marker is intended in part to facilitate in the selection of transformants. Additionally, the presence of such a selectable marker on the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culture medium. In this embodiment, such a pure culture of the transformed host microorganisms would be obtained by culturing the microorganisms under conditions which require the induced phenotype for survival.

D. Vector Assembly

Upon synthesis and/or isolation of all necessary and desired component parts of the above-discussed cloning vectors, the vectors are assembled by methods generally known to those of ordinary skill in the art. Assembly of such vectors is believed to be within the duties and tasks performed by those with ordinary skill in the art and, as such, is capable of being performed without undue experimentation. For example, similar DNA sequences have been ligated into appropriate cloning vectors, as set forth in Maniatis, T., et al. Molecular Clonxng: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1982), which is specifically incorporated herein by reference. E. Host Organism

The vectors and methods disclosed here are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms. Prokaryotes are preferred for cloning of DNA sequences and for expression of the gene. The E. coli strains JM105, JM107 and JM109 (available from Pharmacia), as well as Bacilli, Pseudomonas, and Clostridium species, may be used for expression of the gene. In addition to prokaryotes, eukaryotic microbes, such as Saccharomyces cerevisiae may be used for expression of the gene.

In general, plasmid vectors containing operational elements which are derived from species compatible with the host cells are used. For example, E. coli is typically transformed using pBR322, a plasmid derived from E. coli. The Table 1 below indicates the list of host organisms and the compatible vectors.

The following, noninclusive, list of cloning vectors and operational elements for E . coli and other organisms is believed to set forth vectors which can easily be altered to meet the above-criteria and are therefore preferred for use in the present invention. Such alterations are easily performed by those of ordinary skill in the art in light of the available literature and the teachings herein.

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27. Gray, G.L., McKeown, K.A., Jones, A.J.S., Seeburg, P.H. and Heyneker, H.L. Biotechnology, 161-165 (1984).

28. Lory, S., and Tai, P.C. Gene 22, 95-101 (1983).

29. Liu, P.V. J. Infect. Dis. 130 (suppl), 594-599 (1974).

30. Wood, D.G., Hollinger, M.F., and Tindol, M.B. J. Bact. 145, 1448-1451 (1981).

31. St. John, T.P. and Davis, R.W. J. Mol. Biol. 152, 285-315 (1981).

32. Hopper, J.E., and Rowe, L.B. J. Biol. Chem. 253, 7566-7569 (1978).

33. Denis, C.L., Ferguson, J. and Young , E . T . J . Biol . Chem . 2 58 , 1165-1171 ( 1983 ) .

34. Lutsdorf, L. and Megnet, R. Archs. Biochem. Biophys. 126, 993-944 (1968).

35. Meyhack, B., Bajwa, N., Rudolph, H. and Hinnen, A. EMBO. J. 6, 675-680 (1982).

36. Watson, M.E.E. Nucleic Acid Research 12, 5135-5164 (1984).

37. Gerband, C. and Guerineau, M. Curr. Genet. 1, 219-228 (1980).

38. Hinnen, A., Hicks, J.B. and Fink, G.R. Proc. Natl. Acad. Sci. USA 75, 1929-1933 (1978).

39. Jabbar, M.A., Sivasubramanian, N. and Nayak, D.P. Proc. Natl. Acad. Sci. USA 82, 2019-2023 (1985).

The Examples which are described below use E. coli using the lactose promoter system or the tac promoter system, and the beta-lactamase or the ompA signal sequences. However, it would be well within the abilities of one skilled in the art to construct an expression vector which expresses a.nd secretes HPSTI from alternative prokaryotic or eukaryotic host organisms by analogous techniques.

It is to be understood that additional cloning vectors may now exist or will be discovered which have the above-identified properties and are therefore suitable for use in the present invention . These vectors are also contemplated as being within the scope of the disclosed series of cloning vectors into which the portable DNA sequences may be introduced, along with any necessary operational elements, and which altered vector is then included within the scope of the present invention and would be capable of being used in the recombinant-DNA method set forth more fully below. III. Recombinant DNA Method for Producing HPSTI and HPSTI Analogs

This invention also relates to a recombinant-DNA method for the production of HPSTI and HPSTI analogs. Generally, this method includes:

(a) preparation of a portable DNA sequence capable of directing a host microorganism to produce a protein having trypsin-inhibiting activity;

(b) cloning the portable DNA sequence into a vector capable of being transferred into and replicating in a host microorganism, such vector containing operational elements for the portable DNA sequence;

(c) transferring the vector containing the portable DNA sequence and operational elements into a host microorganism capable of expressing the human pancreatic secretory trypsin inhibitor or an analog thereof;

(e) harvesting the inhibitor in an active form.

In this method, the portable DNA sequences are those synthetic or naturally-occurring polynucleotides described above in Section I. In a preferred embodiment of the present method, the two protable DNA sequences specifically set forth in Section I are employed.

The vectors contemplated as being useful in the present method are those described above in Section II. In a preferred embodiment, the E. coli expression vectors pSGEl and pSGE9 are used in the disclosed method.

The vector thus obtained is then transferred into the appropriate host microorganism. It is believed that any microorganism having the ability to take up exogenous DNA and express those genes and attendant operational elements may be chosen. It is preferred that the host microorganism be an anaerobe, facultative anaerobe or aerobe. Particular hosts which may be preferable for use in this method include yeasts and bacteria. Specific yeasts include those of the genus Saccharomyces, and especially Saccharomyces cerevisiae while specific bacteria include those of the genera Bacillus and Escherichia and Pseudomonas. Various other preferred hosts and vectors are set forth in Table I, surpa.

If it is contemplated that the recombinant HPSTI or HPSTI analog will ultimately be expressed in an organism other than E. col, it is preferred that the cloning vector first be transferred into Escherichia coli, where the vector would be allowed to replicate and from which the vector would be obtained and purified after amplification. The vector would then be transferred into the yeast or other organism for ultimate expression of the inhibitor.

After a host organism has been chosen, the vector is transferred into the host organism using methods generally known by those of ordinary skill in the art. Examples of such methods may be found in Advanced Bacterial Genetics by R.W. Davis et al., Cold Spring Harbor Press, Cold Spring Harbor, New York, (1980), which is specifically incorporated herein by reference.

Any conditions necessary for the regulation of the expression of the DNA sequence, dependent upon any operational elements inserted into or present in the vector, would be in effect at the transformation and culturing stages. In one embodiment, the cells are grown to a high density in the presence of appropriate regulatory conditions which inhibit the expression of the DNA sequence. When optimal cell density is approached, the environmental conditions are altered to those appropriate for expression of the portable DNA sequence. It is thus contemplated that the production of the HPSTI or HPSTI analog will occur in a time span subsequent to the growth of the host cells to near optimal density, and that the resultant inhibitor will be harvested at some time after the regulatoryconditions necessary for its expression were induced.

The host microorganisms are cultured under conditions appropriate for the expression of the HPSTI or HPSTI analog. These conditions are generally specific for the host organism, and are readily determined by one of ordinary skill in the art, in light of the published literature regarding the growth conditions for such organisms , for example Bergey ' s Manual of Determinative Bacteriology, 8th Ed., Williams & Wilkins Company, Baltimore, Maryland, which is specifically incorporated herein by reference.

In certain circumstances, the HPSTI or HPSTI analog will assume its proper, active structure upon expression in the host microorganism and transport of the protein through the cell wall or membrane or into the periplasmic space. This will generally occur if DNA coding for an appropriate leader sequence has been linked to the DNA coding for the recombinant protein. The preferred HPSTI and HPSTI analogs of the present invention will assume their mature, active form upon translocation out of the inner cell membrane when the signal peptide as set forth in Example 1 is incorporated into the cloning vector. Moreover, the structures of numerous other signal peptides have been published, for example by Marion E.E. Watson in Nuc. Acid Res. 12:5145-5164 (1984), specifically incorporated herein by reference. It is intended that these leader sequences, together with portable DNA, will direct intracellular production of a fusion protein which will be transported through the cell membrane and will have the leader sequence portion cleaved upon release from the cell.

In a preferred embodiment, the signal peptide of the beta-lactamase protein is used as a leader sequence and is located in a position contiguous with the portable DNA sequence coding for the inhibitor structure. Additionally preferred leader sequences include those of E. coli OmpA protein and that of carboxypeptidase G2. These and other leader sequences are described above.

It is to be understood that application of the teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation and manufacture appear in the following examples.

EXAMPLE 1 The amino acid sequence of human pancreatic secretory trypsin inhibitor (hereinafter referred to as "HPSTI") has been published by Bartelt, D.C., et al., Arch. Biochem. Biophys. 179:189-199 (1977), and by Yamamoto, T., et al., Biochem. and Biophys. Res. Comm. 132:605-612 (1985), both of which are specifically incorporated herein by reference. The present inventors have discovered a gene to instruct E. coli to make this protein, using in part the sequence of Bartelt et al., the standard genetic code and appropriate expression and control elements used by this bacterium. In practice, a limited subset of codons was used that correspond to those used in highly expressed proteins in E. coli, and that provide convenient restriction sites for construction of the gene.

The upper row of the DNA sequence chosen to code for the mature HPSTI is:

GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC GGT TGC ACT AAA ATC TAC AAC CCG GTA TGT GGT ACC GAC GGT GAC ACC TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT CAG ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

To terminate translation, the codon TAA was added to the end of the DNA. To facilitate cloning of the DNA, a Sall site was added after the TAA.

To start translation, and to provide an amino acid sequence capable of translocating the protein to the periplasmic space of E. coli., the following sequence of DNA was added:

Start of

Translation

10 20 30 40 50

AATTCAGAT CTAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCGC

60 70 80 90

TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGCT

An EcoRI restriction site is included in the DNA to facilitate cloning of the DNA into a range of E. coli. plasmids.

The DNA whose structure is described above was synthesized from the following oligonucleotides prepared according to manufacturers' recommendations in the ABI 380A DNA Synthesizer.

1. AATTCAGATCTAAGGAAGAGTATGAGTATT

2. CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT

3. TGCCTTCCTGTTTTTGCTGACTCT

4. CTGGGTCGTGAAGCTAAGTGCTACAACGAA

5. CTGAACGGTTGCACTAAAATCTACAACCCG

6. CATACTCTTCCTTAGATCTG

7. CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT

8. AGCAAAAACAGGAAGGCAAAATGC

9. GTAGCACTTAGCTTCACGACCCAGAGAGTC

10. GATCCGGGTTGTAGATTTTAGTGCAACCGTTCAGTTCGTT

11. GATCCCGGTATGGTACCGACGGTGAC

12. ACCTACCCGAACGAATGCGTGCTGTGCTTCGAA

13. AACCGTAAACGTCAGACCTCCATCCT

14. GTAGGTGTCACCGTCGGTACCACATACCGG

15. ACGGTTTTCGAAGCACAGCACGCATTCGTTCGG

16. GATCAGGATGGAGGTCTGACGTTT

17. GATCCAGAAATCTGGTCCGTGCTAAG

18. TCGACTTAGCACGGACCAGATTTCTG Oligonucleotides were purified by gel electrophoresis and 5' phosphorylated before use.

The following oligonucleotides were combined pairwise, heated in a water bath to 90°C and allowed to cool slowly: 1. and 6.; 2. and 7.; 3. and 8.; 4. and 9.; 5. and 10. After the pairs were combined, EcoRI and BamHI cut M13 mp18 or mp19 was added and these materials were ligated according to standard procedures as set forth in Maniatis, T., et al., Molecular Clonic - A. Laboratory Manual, Cold Spring Habor Laboratory (1982), specifically incorporated herein by reference. The resulting mixture was used to transform E. coli JM105 and plaques containing the DNA of interest were selected by their ability to hybridize to the labelled oligonucleotides used in the construction. The sequence of the DNA between the EcoRI and BamHI sites was determined by the "dideoxy" sequencing protocol as set forth by Sanger, F., et al., in Proc. Natl. Acad. Sciences 74:5463-67 (1977), specifically incorporated herein by reference. The DNA was prepared from the cells according to standard procedures and cleaved with EcoRI and HpaII restriction endonucleases. A 152-base pair fragment of DNA was purified from the digest by electrophoresis. This sequence is referred to as "Fragment One."

The following oligonucleotides were combined pairwise, heated in a water bath to 90°C and allowed to cool slowly: 11. and 14.; 12. and 15.; 13. and 16.; 17. and 18. After the pairs were combined, BamHI and Sail cut M13 mpl8 or M13 mpl9 was added and these materials were ligated according to standard procedures reported by Maniatis, as set forth above. The resulting mixture was used to transform E. coli JM105 and plaques containing the DNA of interest were selected by their ability to hybridize to the labelled oligonucleotides used in the construction. The sequence of the DNA between the BamHI and Sall sites was determined by the "dideoxy" sequencing protocol as set forth above. The DNA was prepared from the cells according to standard procedures, and cleaved with Sall and Hpall restriction endonucleases. A 109-base pair fragment of DNA was purified from the digest by gel electrophoresis. This fragment, hereinafter referred to as "Fragment Two," is bounded by Hpall and Sall sticky ends.

Fragment One and Fragment Two were mixed with M13 mp18 or M13 mpl9 DNA that had been cleaved with EcoRI and Sall endonucleases and ligated according to standard protocols. The DNA was transformed into E. coli JM105 and DNA was isolated from a plaque which hybridized to selected oligonucleotides used in the construction. This DNA was cut with EcoRI and PstI and ligated with pUC8 cut with EcoRI and PStl. The resulting DNA was transformed into E. Coli JM105.

Ampicillin-resistant colonies were examined for production of material that cross-reacted with rabbit antibodies to HPSTI when grown in the presence of isopropyl thiogalactoside. Several such colonies were grown up, and plasmid structure was verified by restriction site analysis. One plasmid structure was verified by restriction site analysis. One plasmid with the correct structure was designated pSGEl, and the E. coli strain JM105/pSGEl (subsequently referred to as SGE3 ) was picked for further examination.

The chosen clone was grown in LB medium containing ampiciilin at 50ug per ml; 0.2mM IPTG was added at 0.2 A600/ml and growth continued to 0.7 A600/ml. 50ml of the culture broth were centrifuged and the cell pellet was washed in 50mM Tris.HCl, pH 7.5, containing 20% sucrose. The washed cells were resuspended in the same solution and treated with 0.5mg per ml lysozyme in 20mM EDTA for 5 min at 0°C. The solution was again centrifuged for 5 min at 10,000 g and the supernatant, which contained the periplasmic contents of the cells, was analysed by chromatography.

One tenth the initial volume of 1% trifluoroacetic acid (TFA) in water was added and 50ul of the supernatant was applied to a Synchropak RP8 reverse phase high pressure liquid chromotography (hplc) column. The column was eluted with 1 ml/min. of a linear gradient of 0.1% TFA in acetonitrile increasing from 0 to 50% at 1% per minute. A peak of material that absorbed light of 215 and 280nm was detected at 24% acetonitrile. This material inhibited trypsin-catalysed hydrolysis of carbobenzoxy arginine p-nitroanilide. Material from the peak was analysed on an ABI protein sequencer and had the sequence:

Asp Ser Leu Gly Arg Glu Ala Lys X Tyr Asn

Gin Leu Asn Gly Y Thr Lys Z Tyr Asn in which all identified residues are identical to those of the first 20 amino acids of isolated HPSTI and the presence of the unknown residues X and Y is attributed to their presence in S-S bonds. The results described indicate that a gene has been constructed that, when expressed in E. coli., leads to the appearance in the periplasmic space of a protein which (a) co-chromatographs with native HPSTI, (b) reacts with antibodies to HPSTI, (c) has the first 20 amino acids characteristic of HPSTI, and (d) has anti-trypsin activity. These procedures were therefore sufficient to produce an active protein that is identical to a human protein.

EXAMPLE 2 On the basis of the amino acid sequence of Bartelt et al., and of amino acid sequence predicted from the cDNA sequence of Yamamoto et al., the codon usage in highly expressed genes of Escherichia coli, and the provision of convenient restriction endonuclease cleavage sites, the following DNA sequence for HPSTI was proposed:

GACTCTCTGG GTCGTGAAGC TAAGTGCTAC AACGAACTGA ACGGTTGCAC TAAAATCTAC

GACCCGGTCT GCGGTACCGA TGGTAACACC TACCCGAACG AATGCGTGCT GTGCTTCGAA AACCGTAAAC GTCAGACCTC CATCCTGATC CAGAAATCTG GTCCGTGCTA A

To regulate the expression of the protein in a form suitable for export to the periplasm of E. coli, the following regulatory elements are proposed: a tac promoter on plasmid pKK223-3 for initiation of transcription at high levels; a lac repressor (lacI^q), to be encoded on the chromosome of E. coli. strain JM109; an OmpA Shine-Dalgarno sequence to initiate translation at a high level; an OmpA leader to facilitate periplasmic export of the product; an Ala of an Ala-Asp junction between the protein sequence encoded by these operator elements and that encoded by the structural genes described above to dictate cleavage of the initial product to yield the mature HPSTI. The OmpA elements are incorporated in to the following DNA sequence:

GAATT CGATA TCTCG TTGGA GATAT TCATG ACGTA TTTTG GATGA TAACG AGGCG CAAAA AATGA AAAAG ACAGC TATCG CGATC GCAGT GGCAC TGGCT GGTTT CGCTA CCGTA GCGCA GGCT. A. Construction of Gene Fragments

To construct the above OmpA sequence, the following deoxyribonucleotides are synthesized using the ABI DNA synthesizer (Foster City, California). The products are purified by polyacrylamide gel electrophoresis as described in the ABI instrument manual. They are 5' phosphorylated using T4 polynucleotide kinase and ATP using standard means.

The following group of oligonucleotide sequences are used to construct fragment Aa. Oligonucleotide Aa1 is:

AATTCGATATCTCGTTGGAGATATTCATGACGTATTTTGGATGATAACGAGGCGCAAAAA. Oligonucleotide Aa2 is: ATGAAAAAGACAGCTATCGCGATCG. Oligonucleotide Aa3 is:

GATCCGATCGCGATAGCTGTCTTTTTCATTTTTTGC. Oligonucleotide Aa4 is:

GCCTCGTTATCATCCAAAATACGTCATGAATATCTCCAACGAGATATCG. Oligonucleotide Aa5 is:

GATCCGATCGCAGTCGCACTGGCTGGTTTCGCTAGCGCAGGCCTCTGGTAAA. Oligonucleotide Aa6 is: AGCTTTTACCAGAGGCCTGCGCTACGGTAGCGAAACCAGCCAGTGCCACTGCGATCG.

The following groups of oligonucleotides are mixed and annealed under standard conditions and ligated to each other and to cloning and sequencing vectors M13 mp18 and M13 mp!9 cut with appropriate restriction endonucleases using T4 DNA ligase under standard conditions. The products are used to transform E . coli JM105, and clones containing the DNA of interest are selected by hybridization with 32p labeled oligonucleotides selected from the group used in the annealing step. The insert structure is confirmed by dideoxy sequencing of the cloned DNA using a universal primer.

Oligonucleotides Aal-Aa4 are ligated to M13 mp18 and M13 mp19 cut with EcoRI and BamHI. M13 replicative-form DNA having the desired insert DNA is recovered by standard means. The insert DNA is excised from the M13 DNA by cutting the M13 DNA with restriction endonucleases EcoRI and Pvul and is purified by polyacrylamide gel electrophoresis. Its structure is: AATTCGATATCTCGTTGGAGATATTCATGACGTATTTTGGATGATAACGAGGCGCAAAAAATGA

GCTATAGAGCAACCTCTATAAGTACTGCATAAAACCTACTATTGCTCCGCGTTTTTTACT AAAAGACAGCTATCGCGAT TTΓICTGTCGATAGCGC

Oligonucleotides Aa5 and Aa6 are ligated to M13 mp18 and M13 mp19 cut with BamHI and Hindlll. M13 replicative-form DNA having the desired insert DNA is recovered by standard means. The insert DNA is excised from the M13 DNA by cutting the DNA with restriction endonuclease Pvul and Hindlll and is purified by polyacrylamide gel electrophoresis. Its structure is:

CGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCTCTGGTAAA TAGCGTCACCGTACCGACCAAAGCGATGGCATCGCGTCCGGAGACCATTTTCGA This Pvul-Hindlll fragment is combined with the EcoRI-PvuI fragment prepared from oligonucleotides Aal-Aa4 and ligated with M13 mp18 or M13 mp19 cut with EcoRI and Hindlll. M13 replicative-form DNA having the desired insert DNA is recovered by standard means. The insert DNA, which is DNA fragment Aa, is excised from the M13 DNA by cutting the M13 DNA with restriction endonucleases EcoRI and Hindlll. Its structure is: AATTCGATATCTCGITGGAGATATTCATGACGTATTTTGGATGATAACGAGGCGCAAAAAATGA

GCTATAGAGCAACCTCTATAAGTACTGCATAAAACCTACTATTGCTCCGCGTTTTTACT AAAAGACAGCTATCGCGATCGCAGTGGCACTGGCTGGTTCGCTACCGTACGCGCAGGCCTCTGGTA TTTTCTGTCGATAGCGCTAGCGT CACCGTACCGACCAAAGCGATGGCATCGCGTCCGGAGACCAT AA

TTTCGA

The DNA was further digested with Clal and PvulI in order to destroy the M13 vector DNA. The reaction was phenol extracted, and the DNA was ethanol precipitated. After pelleting, the DNA was dissolved, mixed with EcoRI/ Hindlll-cut pUC8, and ligated.

E. coli Jm109 was transformed with this DNA, and ampicillin-resistant colonies were screened by hybridization to labeled oligonucleotide probes, and plasmid structure was checked by restriction site mapping.

One clone with the correct structure, pOmpS.S.11-C, was altered as follows to allow fusion of the OmpA signal sequence to HPSTI. pOmpS.S.11-C was cut with StuI and Hindlll, and a phosphorylated oligonucleotide adaptor with the sequence: p-CTGACTCTGGTAAA

GACTGAGACCATTTTCGA-p was ligated to the Stul/HindIll-cut plasmid, which was used to transform JM109.

Transformants were screened by hybridization to labeled oligonucleotide, and plasmid structure was checked by restriction site analysis. The plasmid was cut with EcoRI and Hinfl and a 123-bp fragment was isolated. This was mixed with a Hinfl/Sall fragment from plasmid. pSGEl, described in an earlier example, and the two fragments ligated to M13 mp18 cut with EcoRI and Sall. Transformants were screened by hybridization, and for several clones, the DNA sequence containing the flanking the Hinfl site was determined to verify their structures. One clone, OmpA-HPSTI:mp18, was picked for further work.

Mutagenesis of OmpA-HPSTI:mp18 was performed using standard oligonucleotide-directed, site-specific mutagenesis as described in Zoller, M., Jr., and Smith, M., Methods in Enzymology Voi. 100, page 468 (1983); Kunkel, T.A. Proc. Natl. Acad. Sci. 82:488 (1985), specifically incorporated herein by reference. The sequence of the mutagenic oligonucleotide was:

GGTGTTACCATCGGTACCGCAGACCGGGTCGTAGAT

A clone, OmpA-HPSTI-m:mp18, was chosen by hybridization screening (using the mutagenic oligonucleotide as the probe) and DNA sequencing. RF DNA derived from this clone was cut with EcoRI and PstI; the 295-bp fragment was purified and ligated with EcoRl/Pstl-cut pkk223-3. The resulting DNA was used to transform JM109.

Ampicillin-resistant colonies were examined for production of material that cross-reacted with rabbit antibodies to HPSTI when grown in the presence of isopropyl thiogalactodside. Several clones were grown up, and plasmid structure was checked by restriction site analysis. One plasmid with the correct structure was designated pSGE9, and the pSGE9 containing E. coli strain, JM109, is designated SGE45.

The chosen strain, SGE45, was grown in LB medium containing ampiciilin at 50ug per ml at 37°C. At a cell density of 0.15 A_660/ml, IPTG was added to 0.5 mM concentration and the cells allowed to grow to 30 A₆₆₀/ml. About 1 ml of culture broth was centrifuged at the various times after induction, and medium was saved for assaying HPSTI activity. Figure 3 shows the growth curve of SGE45 and HPSTI activity found in the medium.

At a A_{66 0}/ml of 30, 5 liters of the culture broth was centrifuged at 8000g for 20 minutes to remove cells, and the HPSTI in the medium was purified by affinity chromatography on a column of 20 mg bovine trypsin bound to 1 ml of affigel-10. The 200 ml of this affinity resin absorbed about 1 g of HPSTI. Bound HPSTI was eluted with 50 mM HCl. Minor contaminating proteins in the above affinity-purified HPSTI preparations were removed by the linear gradient of NaCl [0 -0.1 M] on SP-C25 ion-exchange chromatography [25 x 25 cm]. In addition, these columns could be run in reverse order. From 5 liters of culture medium,, 0.7 g of HPSTI was purified. About 2 nmoles of this material was analyzed without reduction-carboxymethylation on an ABI protein sequencer and had the sequence: Asp Ser Leu Gly Arg Glu Ala Lys X Tyr Asn in which unknown residue X is supposed to be Cys. This sequence is identical to the natural sequence that was published by Bartelt, supra, and Yamamoto et al., supra.

It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS

1. A human pancreatic secretory trypsin inhibitor comprising a protein possessing at least one active site with the ability to inhibit proteases, wherein said inhibitor is substantially homologous to that isolatable from human pancreatic tissues and wherein said inhibitor is produced using recombinant DNA methods.

2. The human pancreatic secretory trypsin inhibitor of claim 1 wherein said inhibitor is encoded by the following DNA sequence:

GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC

GGT

TGC ACT AAA ATC TAC AAC CCG GTA TGT GGT ACC GAC GGT GAC

ACC

TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT

CAG

ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

3. The human pancreatic secretory trypsin inhibitor of claim 1 wherein said inhibitor is encoded by the following DNA sequence:

GAC TCT CTG GGT CGT GAA GCT AAG TGC TAC AAC GAA CTG AAC

GGT

TGC ACT AAA ATC TAC GAC CCG GTC TGC GGT ACC GAT GGT AAC

ACC

TAC CCG AAC GAA TGC GTG CTG TGC TTC GAA AAC CGT AAA CGT

CAG

ACC TCC ATC CTG ATC CAG AAA TCT GGT CCG TGC

4. A method for the production of a human pancreatic secretory trypsin inhibitor comprising:

(a) preparation of a portable DNA sequence capable of directing a host microorganism to produce an inhibitor protein that is substantially homologous to the human pancreatic secretory trypsin inhibitor isolatable from human pancreatic tissues;

(c) transferring the vector containing the portable DNA sequence and operational elements into a host microorganism capable of expressing said inhibitor protein;

(d) culturing the host microorganism under conditions appropriate for amplification of the vector and expression of the inhibitor protein; and

(e) harvesting the inhibitor protein in an active form.

5. The method of claim 4 wherein said inhibitor protein is human pancreatic secretory trypsin inhibitor.

6. The method of claim 4 wherein said inhibitor protein is an analog of human pancreatic secretory trypsin inhibitor.