WO1991014780A1

WO1991014780A1 - Yeast expression vectors

Info

Publication number: WO1991014780A1
Application number: PCT/US1991/001831
Authority: WO
Inventors: Pudur Jagadeeswaren
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1990-03-21
Filing date: 1991-03-20
Publication date: 1991-10-03
Also published as: AU7586991A

Abstract

The expression of polypeptides, in particular biologically functional human placental factor XIII, in a novel yeast expression vector at levels which constitute as much as 2 % protein of the partially purified cell-free extract is disclosed in the present invention. The advantages of the expression vector of the invention are demonstrated using Factor XIII which is the last zymogen to be activated in the vertebrate blood coagulation cascade. The active form, Factor XIIIa, is a transglutaminase that covalently cross-links fibrin molecules by joining gamma-glutamyl and ε-lysyl primary amine groups. Prior to the present invention, this important blood coagulation factor was derived from human sera with the potential for contamination inherent in that process.

Description

YEAST EXPRESSION VECTORS

The National Institutes of Health provided funding used in part for this invention under grant HL36226. Accordingly, the Federal Government may have certain rights in this invention pursuant to 35 U.S.C 202.

The invention relates to yeast expression vectors and to yeast host cells containing such vectors for expressing polypeptides. The invention relates further to a process for constructing a yeast expression vector which vector comprises a DNA fragment containing the protein in frame with a high-level expression promoter derived from yeast which promoter sequence is contained in the multiple cloning region of a yeast cloning vector. More specifically- the invention relates to the expression of a blood coagulation factor using such an expression vector in a yeast host cell, in particular the blood coagulation Factor XIII.

The baker's yeast Saccharomγces cerevisiae (S. cerevisiae) has been used successfully as a host for heterologous protein synthesis. As a host, this yeast is preferred over other yeasts and fungi due to the fact that it is not a pathogen and that it is widely accepted as a fermentation organism in the food and pharmaceutical industries. Unlike E. coli, yeasts produce no endotoxins; therefore, adequate purification of pharmaceutical products is likely to be more easily accomplished.

From a molecular biological standpoint, yeasts exhibit desirable characteristics as host cells for expression of protein. In particular, yeasts exhibit post-translational modification systems (glycosylation, acetylation, phosphorylation, etc.) which are lacking in prokaryotic host cell systems. The ability to introduce foreign DNA into yeast cells and into the yeast genome has lead to the expression of a number of eukaryotic proteins in S . cerevisiae (Broker and Bau l 1989) .

With the advent of methods which allowed the transformation of yeast host cells (as spheroplasts) and the recognition that certain genes from yeasts could be used to complement bacterial mutations in analogous bacterial genes, it has been possible to construct shuttle cloning vectors. These vectors are essentially plas ids which contain both bacterial sequences that signal DNA replication in E. coli and sequences that signal DNA replication in yeast. In addition, in order to allow effective selection in both bacteria and yeast, these shuttle vectors usually contain a selectable marker for a bacterial antibiotic resistance factor (e.g., for ampicillin resistance) to enable selection for the plasmid in bacteria as well as a yeast biosynthetic gene (e.g., for complementing a leucine biosynthetic gene mutation of the host yeast cell) to enable selection for the plasmid in yeast.

Host strains of yeast contain an autonomously replicating ring of DNA called the 2μ circle. This yeast plasmid contains about 6300 base pairs of DNA and encodes a single yeast origin of replication. In addition, the 2μ yeast plasmid encodes two replication functions that promote amplification of the 2μ circles when the copy number is low. These sequences are required on yeast cloning vectors since they insure that the single plasmid, once transformed, will be brought back up in copy number to around 30-40 copies per cell.

In addition, sequences are typically incorporated into yeast cloning vectors which allow the plasmid to autonomously replicate without a requirement for integration into the yeast chromosome. Such sequences (called autonomously replicating sequence, or ARS) have been isolated from yeast sources and contain at least 60 base pairs of DNA. An ARS is required in a yeast cloning vector in order to insure that the plasmid DNA will not incorporate into the host cell's chromosome, that the plasmid will remain an episome and will thereby be amplified in copy number.

Even when bacterial plasmids containing inserted ARS elements and foreign genes are used to transform yeast cells, and even though the efficiency of transformation is high, the plasmid is typically lost from these cells as they multiply and the selective pressure is removed (e.g., after ten generations, typically only 5% of the cells still contain the plasmid) . This is due to the fact that during cell division, the plasmids apparently do not segregate regularly and uniformly between the two daughter cells. Additional yeast DNA sequences have been found necessary to insure uniform segregation during meiosis. DNA segments containing sequences from the centromere (CEN) regions of yeast chromosomes are, therefore, typically introduced into such vectors to stabilize their presence in succeeding generations of daughter cells.

Therefore, prior investigations have established that efficient plasmid vectors designed specifically for yeast transformat-ion should contain: sequences of a bacterial plasmid containing both the bacterial origin of replication and a selectable antibiotic resistance factor, an ARS sequence, a CEN sequence, a selectable yeast marker gene, and one or more unique restriction sites to allow insertion of the heterologous DNA (Watson et al. 1983) . The variously required sequences invariably add to the overall size of any shuttle vector that is to be genetically manipulated in the bacterial host.

As plasmids reach approximately 10,000 to 15,000 base pairs in size, transformation efficiency is drastically reduced in most bacteria (Maniatis 1982) . Thus, one desiring to construct a yeast cloning vector must balance the need for the various sequences described above with the need to limit the overall size of the vector. This balancing requirement is exacerbated considerably when large sequences of heterologous DNA are inserted into such cloning vectors.

Additionally, where one wishes to efficiently express a foreign protein in yeast, unless the heterologous gene to be expressed in yeast carries its own promoter sequence which is itself functional in yeast, additional DNA sequences must be added to the cloning vector to promote expression of the heterologous gene. Such vectors which contain additional promoter sequences are known as expression vectors. While certain of these vectors and the use of such vectors to express certain proteins have been described in the past, typically they contain sequences which add considerably to the size of the plasmid.

For instance, an expression vector for yeast was described by Mackay, et al. (European Patent Application

No. 314096, published May 3, 1989), where yeast cells were transfor ed with two DNA constructs. The second DNA construct comprised a promoter followed downstream by a DNA sequence encoding a secretional signal and a DNA sequence encoding a heterologous protein. Among those proteins described in combination with this system are several blood coagulation factors, including Factor XIII.

Broker and Bauml (1989) studied expression vectors i a fission strain of yeast Schizosaccharomvces pombe. whic is taxonomically and evolutionarily very distant from baker's yeast. They reported low rates of expression whe promoters from baker's yeast were used in the fission yeast, presumably because transcription signals recognize by the distantly related yeasts were not identical. Thes workers were able to achieve better expression, however, using a promoter homologous in both yeasts, namely the promoter element from the alcohol dehydrogenase gene (adh) . When adh promoter was combined in a yeast cloning vector capable of stable amplification in the fission yeast, and the gene for Factor XIII was placed under the control of this promoter, the heterologous protein was expressed although the level of expression was not disclosed.

Other promoters functional in baker's yeast have bee reported as well. Johnston & pavis (1984) isolated the GAL1-GAL10 promoter region from yeast and constructed a yeast expression vector (pBM150) which contained many of the sequences described as necessary above. The cloning vector from which the expression vector was derived, was an autonomously replicating, centromere-containing plasmi which carried the ura3 gene for selection in yeasts. The GAL1 and GAL10 genes of yeast encode enzymes responsible for galactose utilization in S. cerevisiae and are clustered together on the yeast chromosome. These two genes are adjacent and are transcribed in opposite -6-

directions with approximately 600 base pairs of DNA separating their transcription initiation sites. The authors of this article noted that these GAL promoters are capable of high-level, regulated gene expression and that several genes under GAL1 and GAL10 control successfully expressed their gene products. Deletion mutations which alter the region between these two promoter sequences (the GAL1-GAL10 control region) are described which considerably reduce the amount of DNA necessary to.add to the plasmid, yet which still provide high level expression of heterologous genes.

Other expression vectors incorporating GAL promoter sequences have also been described. Goff et al. (1984) have constructed vectors specifically designed for the expression of calf prochymosin in Saccharόmyces. thus, this vector was not designed as a general cloning vector with a multiple cloning region. These workers found further that it was preferable to add additional sequences to the vector following the prochymosin-encoding DNA. This was accomplished in order to overcome the lack of suitable yeast transcription termination signals in the expression vectors other than those fortuitously present downstream of the cloned gene in the yeast biosynthetic gene DNA or the yeast 2μ DNA contained on the expression vector. Similarly, Miyajima and co-workers (1984) constructed yeast expression vectors lacking a multiple cloning region but which incorporated either GAL promoter sequences or adh promoter sequences. An additional 390 bp fragment carrying the yeast TRP5 terminator was placed downstream of the cloned cDNA fragment.

As noted above, a number of eukaryotic proteins which require correct post-translational processing in order to become active have been coupled to yeast expression vectors. One such protein is Factor XIII which is a -7-

glycoprotein that has been isolated and characterized from plasma, platelets, and placenta. Placental and platelet forms of Factor XIΪI are identical (Bonn 1978) . Both have a subunit structure denoted A₂, which represents the noncovalent association of two identical 83-kDa polypeptides. Plasma Factor XIII similarly contains two A subunits, but is also noncovalently associated with two noncatalytic 85-kDa B subunits (Chung et al. 1974) . The a ino acid sequences of both the A and B subunits (Takahashi et al. 1986; Ichinose et al. 1986a,b) have been deduced, and their corresponding σDNAs have been isolated (Grundmann et al. 1986; Ichinose et al. 1986a,b) .

Workers have attempted to express this important protein using a plasmid which directs the synthesis of the human placental Factor Xllla protein in the unfused form in bacterial host cells. While the protein thus expressed constitutes 2% of the total cellular protein, it is apparently expressed intracellularly in a denatured, biologically inactive form (Amann et al. 1988) .

With increasing concern regarding the sources of blood factors to be administered to human patients, in particular factors which currently must be derived from human serum such as Factor XIII, the production of factors is a favorable alternative source since it allows the development of safer substitution therapy. One of the best hosts from which to derive such blood coagulation factors is the standard yeast used widely in fermentation industries. Yeast expression vectors are needed which balance all of the requirements stated above. The vector should possess stability by incorporating all necessary sequences and yet remain capable of incorporating large DNA sequences typical of complex eukaryotic proteins. The necessity for stabilizing the plasmid in the yeast host cell, for including bacterial sequences for selection and replication in bacteria, and for minimizing the size of the vector in order to maintain its transformation efficiency most preferably must be balanced with the additional requirement for providing high-level expression of the protein in the standard yeast strains of fermentation industries. In particular, such vectors are needed to provide alternate sources for critical blood coagulation proteins such as Factor XIII.

The present invention relates to a combination of a DNA fragment encoding a protein with a high-level yeast promoter and incorporation of this transcriptional and translational unit in a stable yeast cloning vector. The resulting protein derived from yeast host cells is, in a preferred.embodiment, biologically functional and immunologically cross-reactive. It will be understood by those of skill in the art that the compositions of the present invention (including DNA fragments, polypeptides, vectors and host cells) are the result of recombinant DNA technologies and, as such, may properly be referred to as, for example, recombinant polypeptides. For the purposes of this invention, however, the modifier "recombinant" will not be used in each case. In a preferred embodiment, the amount of the protein recovered from the yeast host cells is in a commercially feasible range of approximately 1-2% of the partially purified cell-free extract produced by these cells. A method for constructing the vector of the invention is also disclosed.

More particularly, in a preferred embodiment, the present invention combines a full length cDNA fragment encoding a human blood coagulation factor with a high- level yeast promoter and incorporates this transcriptional and translational unit in a stable yeast cloning vector. The resulting protein derived from yeast host cells is biologically functional and immunologically cross-reactive and the amount of the protein recovered from the yeast host cells is approximately as much as 2% of the protein of the partially purified cell-free extract.

The present invention generally relates to the vectors, DNA segments, purified protein, methods of cloning, and host cells necessary to obtain commercial levels of a polypeptide such as a Factor XIII polypeptide The vector of the present invention may be used for the cloning of any number of proteins, however, the blood coagulation factors such as a Factor XIII polypeptide are of particular interest. Accordingly, the present invention concerns, generally, compositions and methods for the preparation of proteins such as a Factor XIII polypeptide of eukaryotic origin. In a preferred embodiment, the Factor XIII polypeptide will be the catalytic subunit of the Factor XIII, Factor Xllla.

In certain general and overall embodiments, the invention concerns vectors and isolated DNA segments encoding a Factor XIII. The DNA segments of the inventio may encode biologically functional equivalent protein or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged.

In the context of the present invention, the term Factor XIII polypeptide is intended to refer to peptides or proteins having the biological and the immunological identity of the a Factor XIII polypeptide of the human placenta. For example, such a polypeptide may be cloned from a cDNA library of human placenta. Generally, a Factor XIII polypeptide of the invention will refer to an amino acid peptide or protein which is substantially the length of the presently known Factor XIII molecules from hu an placenta. However, the invention does not preclude and, in fact enables, preparation or use of shorter or longer peptides or proteins, so long as a peptide or protein has similar in kind biological activity and/or a cross reactive immunological reactivity, for example, as defined by rabbit polyclonal antisera.

In certain general aspects, the invention relates to the preparation and use of DNA segments, including vectors or DNA fragments, having a sequence encoding a Factor XIII polypeptide. For yeast cloning vectors, any number are known in which DNA sequences of the invention may be incorporated. The vector YEp352 has been demonstrated to be of particular value.

In certain embodiments, the vector will contain a substantially purified DNA fragment which encodes at least a useful portion of a Factor XIII polypeptide which includes substantially all of the amino acids of a biologically active Factor XIII, or functionally equivalent amino acids. Vectors and isolated segments may, therefore, variously include the basic Factor XIII coding region itself or may contain coding regions bearing selected alterations or modifications in the basic coding region of a Factor XIII polypeptide. Alternatively, such vectors or fragments may encode larger proteins or peptides which nevertheless include the basic coding region. In any event, it should be appreciated that due to codon redundancy, as well as biological functional equivalence, this aspect of the invention is not limited to the particular DNA sequences used in the present invention.

In certain preferred embodiments, the invention provides a method for producing a yeast expression vector encoding a Factor XIII polypeptide. Using this method one first obtains a DNA fragment encoding a Factor XIII polypeptide. Next, the method of the invention directs one to obtain a fragment of yeast DNA encoding a GAL1- GAL10 promoter region or derivative thereof. Following this step, a yeast cloning vector which contains a multiple cloning region is cleaved by appropriate means in order to allow the insertion of the fragment of yeast DNA encoding the promoter region into the yeast cloning vector so that the GAL1 promoter is in close proximity to the multiple cloning region. Finally, the DNA fragment encoding a Factor XIII polypeptide is inserted into the multiple cloning region of the vector so produced in a manner which insures that the GAL1 promoter is arranged in a transcriptional and translational unit with a Factor XIII polypeptide.

In this manner, a yeast expression vector encoding a Factor XIII polypeptide in a transcriptional and translational unit with the GALl promoter may be constructed where a Factor XIII polypeptide is inserted into a multiple cloning region of the yeast expression vector 3* of the promoter. Vectors such as those of the present invention are useful both as a means for preparing quantities of a Factor XIII polypeptide-encoding DNA itself, and as a means for preparing the encoded protein and peptides. It is contemplated that where Factor XIII polypeptides of the invention are made by recombinant means, one may employ the vectors as either prokaryotic or eukaryotic expression systems and/or as shuttle systems between the two cell types.

Prokaryotic host cells are disclosed in a preferred embodiment of the invention. However, in that prokaryotic systems are usually incapable of correctly processing eukaryotic precursor proteins, and since eukaryotic Factor XIII's are anticipated in a preferred embodiment of the invention, one may desire to express such sequences in eukaryotic hosts. Even where the DNA segment encodes a eukaryotic Factor XIII, it is contemplated that prokaryotic expression will have some additional applicability. Therefore, the invention can be used in combination with vectors which can shuttle between the eukaryotic and prokaryotic cells.

Where expression of a Factor XIII polypeptide in a eukaryotic host is contemplated, it will be desirable to employ a vector such as that of the present invention which incorporates a eukaryotic origin of replication such as those found within the 2μ circle fragment of yeast. Additionally, for the purposes of expression in eukaryotic systems, one will desire to position a Factor XIII polypeptide encoding sequence adjacent to and under the control of an effective eukaryotic promoter such as promoters used in combination with the GAL1-GAL10 containing plasmids described herein. To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5' end of the transcription initiation site of the proper transcriptional reading frame of the protein between about 1 and about 50 nucleotides 3' of or "downstream" with respect to the promoter chosen.

Furthermore, where eukaryotic expression is anticipated, one will typically desire to incorporate into the transcriptional unit which includes -a Factor XIII polypeptide, an appropriate transcriptional termination site unless the DNA fragment incorporating the protein of interest includes such a site. The termination site may be added downstream of the coding sequence. However, in a preferred embodiment of the invention the termination sequence is provided by the 2μ circle DNA of the expression vector of the present invention without the addition of additional termination sequences. Accordingly, in certain preferred embodiments, the vectors of the invention are those where the Factor XIII polypeptide encoding sequence is positioned adjacent to and under the control of an effective promoter. The promoters may be that set of promoters known well to thos of skill in the art where the promoter comprises a GAL1- GAL10 promoter region, the vector being adapted for expression in a yeast host. Alternatively, the promoter may be any one of those of common knowledge to skilled artisans where the promoter is another eukaryotic promoter. An example of such an additional promoter woul be the promoter region for yeast alcohol dehydrogenase gene, adh.

The invention also provides methods for obtaining a variety of host^' cells which incorporate a DNA sequence encoding a Factor XIII polypeptide in the yeast expressio vector of the present invention. The host cell may be either prokaryotic or eukaryotic in nature. In any case, it is understood that the DNA segment encoding a Factor

XIII polypeptide will also possess the regulatory signals functional in the particular host cell.

In certain general aspects, then, a method of producing a Factor XIII polypeptide is provided by the invention. First, one produces a host cell according to the methods and with the compositions of the invention such that the cell so produced is capable of expressing the polypeptide. Next, one cultures the host cell under conditions appropriate for expressing the polypeptide.. Finally, the polypeptide is recovered.

Pig. l. Construction of factor XIII cDNA in a yeast expression vector. Lambda 13-24 is a full-length cDNA derived by joining lambda 13B-96 [subcloned from lambda 13B(H)] and lambda 13A-18 [subcloned from lambda 13A(I)]. Restriction sites for BamHI, Bσlll . EcoRI, Hindlll, PstI, Sphl. and Xbai are denoted BI, BII, RI, HIII, PI, SI and XI, respectively; * denotes polymorphism of the EcoRI site. pPH4 contains the GAL1-GAL10 promoter, followed by Pstl-PstI (2.5 kb fragment), encoding factor XIII placed into the multiple cloning site (BI, XI, PI, SI and HIII) , URA-3 selectable marker, 2μ circle and ampicillin resistance gene (Amp.. The GAL1 promoter initiates the transcription 56 bp upstream from the BamHI site of the multiple cloning site. The initiation codon of factor

XIII cDNA is located 18 bp downstream from the first PstI site. The stop codon is situated 114 bp upstream from the second PstI site. The transcription termination signal for yeast resides in the 2μ circle.

Fig. 2. Expression of factor XIII.

(A) Factor XIII Western blot. Lane 1, cell-free extract from yeast transformed with pPH3 (ATCC 40777) (4.7 μg total protein) ; lane 2, same as lane 1, but digested with 0.5 units of thrombin; lane 3, human placental factor XIII (FXIII) (0.03 units, Calbiochem Biochemicals) ; lane 4, human placental FXIII (0.03 units) digested with 0.3 units thrombin; lane 5, cell-free extract from yeast transformed with pPH4 (ATCC 40778) (12.9 μg total protein); lane 6, same as lane 5, but digested with 0.5 units thrombin; molecular weight markers (BRL) . Numbers to right indicate the size (in kDa) of markers.

(B) Factor XIII .assays. Lane 1, 0.2 mg human fibrinogen (Calbiochem); lane 2, factor XIII (FXIII) assay mixture without FXIII added; lane 3, FXIII assay mixture with 0.05 units FXIII added. This lane displays typical species from top to bottom: gamma-chain dimers (gamma-gamma) , α- chains (α) , jθ-chains (β) and gamma-chains (gamma) of fibrin; lane 4, FXIII assay mixture with yeast extract from pPH3 transformant; lanes 5 and 6 contain FXIII assay mixtures containing yeast extracts from pPH4 transformants; lane 7, molecular weight markers (BRL) . Numbers to right indicate the size (in kDa) of markers.

In the final stages of blood coagulation, thrombin is produced which activates Factor XIII to the 79-kDa Factor Xllla enzymatic form by removing the amino terminal 36 amino acids (Takagi et al. 1974) . A second thrombin cleavage eventually inactivates Factor Xllla producing a 56-kDa and a 24-kDa fragment (Takahashi et al. 1986) . Factor XIIla is a transglutaminase that forms inter olecular gamma-glutamyl-e-lysyl isopeptide bonds between fibrin molecules. This cross-linking results in a fibrin clot of significant mechanical strength and confers resistance to proteolytic breakdown by plasmin.

Deficiencies of Factor XIII result in prolonged bleeding, defective wound healing, and habitual abortions. Congenital deficiencies occur with a frequency of 1 in 2 X 10⁶ individuals and are treated by substitution therapy. Acquired Factor XIII deficiencies occur in patients suffering from leukemia (Egbring et al. 1977), uremia (Nussbaum and Morse 1964) , erosive gastritis (Nilsson et al. 1975), Weber-Christian disease (Henriksson et al. 1975), and Henoch-Schonlein purpura (Kimitsuji et al. 1987) ; Factor XIII concentrates aid in treating these disorders. An absence of Factor XIII activity in an individual warrants substitution therapy, and, since the availability of Factor XIII concentrates free of blood contaminants such as AIDS and hepatitis viruses is limited, the production of a factor XIII as provided by the methods and compositions of the present invention is desirable.

While numerous constructions were available which incorporated a Factor XIII from some DNA source into cloning vectors, including attempts by the present inventor and others to express the protein in yeast, the present inventor sought a readily manipulatable vector incorporating the protein. In order to achieve this end, the present inventor had to obtain independently a full length cDNA encoding human placental Factor XIII. The present inventor then attempted to express this protein in a variety of standard vectors under the control of a variety of standard promoter systems with no success. For instance, the present inventors attempted to get expression of Factor Xllla under the control of the lacZ promoter in £. coli without success. Similarly, no acceptable expression was observed when the Factor Xllla of the present invention was placed under control of the early promoter of SV40 virus and used in conjunction with mammalian cells.

It was reasoned that the failures experienced by the present inventor were the result of several factors. The most important appeared to be due to the size of the Factor XIII fragment. The size of prior art cloning vectors appeared to limit the efficiency of transformation in the bacterial host. This limitation was critical since all genetic manipulations were first carried out in the bacterial host. This stimulated the present inventor to seek an alternative vector for expression in a eukaryote such as yeast, but which vector would possess not only an ability to accept cloned fragments into a multiple cloning region of unique restriction sites, but also to minimize the ultimate size of the vector with the cloned fragment in order to eliminate transformation efficiency problems. None of the prior art yeast cloning vectors appeared capable of achieving the sought after attributes. Either they lacked a multiple cloning region, were too large to accept large fragments of recombinant DNA while maintaining their ability to readily transform, lacked an efficient yeast promoter sequence or some combination of these deficiencies.

Thus, the present inventor combined the cDNA fragment in a novel yeast expression vector which was of the correct size range and which possessed the necessary stabilizing sequences required of effective yeast cloning vectors. In addition, the present inventor sought a promoter functional in yeast which had the potential for commercial level expression. Surprisingly, when a portion of the yeast vector pBM150 containing the GAL1-GAL10 promoter system was inserted into the multiple cloning region of YEp352 and the cDNA fragment of factor XIII was inserted into the PstI restriction site within that multiple cloning region, a vector was created which incorporated all the aspects the present inventor sought. The vector is much smaller than prior art constructions allowing ease of manipulation in the bacterial intermediate host. The vector is, at the same time, a stable yeast expression system providing a method for producing commercial levels of biologically active, immunologically cross-reactive human placental Factor XIII for use in substitution therapy. Finally, and somewhat surprisingly, no down stream sequences were necessary as was suggested in certain prior art studies for high-level expression, allowing the present inventor to minimize the ultimate size of the vector.

Deposits of the yeast expression vector and the yeast expression vector incorporating the Factor Xllla encoding DNA fragment of certain preferred embodiments of the present invention were transmitted on March 19, 1990 under the directives of the Budapest Treaty to the American Type Culture Collection. Specifically, a vector was transmitted to be deposited with the American Type Culture Collection as ATCC number 40777 (pPH3) and corresponds to the yeast expression vector of a preferred embodiment of the invention. Another vector was transmitted to be deposited with the American Type Culture Collection as ATCC number 40778 (pPH4) and corresponds to the yeast expression vector of a preferred embodiment of the invention and which also contains the Factor Xllla fragment of a preferred embodiment of the invention.

Accordingly, an example utilizing the vector of the invention has been included below in order to illustrate preferred modes of the invention. Certain aspects of the following example are described in terms of techniques and procedures found by the present inventor to work well in the practice of the invention and exemplify the use of standard laboratory practices of the inventor. In light of the present disclosure and the general level of skill in the art, those of such general skill will recognize that the following example pertaining to Factor XIII is intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the invention.

Example I: Synthesis of Human Coagulation Factor XIII in Yeast

The following abbreviations are used in the example below: HRP, horse radish peroxidase; PA, polyacrylamide; pfu, plaque-forming units; SDS, sodium dodecyl sulfate; TBS, 50 M TrisΗCl, pH 8.0, 150 mM NaCl; TBST, -50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Tween 20.

MATERIALS AND METHODS

(a) Factor XIII cDNA cloning and construction of yeast expression vector Clone lambda 13A(I) was obtained by screening approximately 5 X 10⁵ pfu of a lambda gtll human placental cDNA library (Clonetech Laboratories) with a polyclonal anti-factor Xllla antibody (Calbiochem, San Diego CA) as described by Young and Davis (1983). The antiserum was diluted 1000-fold in TBST and used to screen approximately 3 X 10* pfu per 20 cm X 20 cm plate. Positive clones were further purified by two cycles of screening at low plaque densities. A positive phage clone, lambda 13A(I) , was amplified and the DNA was isolated, subcloned, and sequenced by supercoiling (Chen and Seeburg 1985) and automated Maxam and Gilbert methods (Jagadeeswaran and Kaul 1986) . The clone was confirmed to be factor XIII cDNA by nucleotide sequence comparisons with published data (Grundmann et al. 1986; Ichinose et al., 1986a). The 5'-terminal Eco^'RI fragment of lambda 13A(I) was nick- translated and used to rescreen the library as described by Benton and Davis (1977) . The DNA from positive clone lambda 13B(H) was isolated (Maniatis et al. 1982) , subcloned, and sequenced.

The EcoRI fragment of lambda 13B(H) was subcloned into pUCl9 creating lambda 13B-96. The BamHI+Bσlll fragment of lambda 13A(I) was subcloned into the BamHI site of pUC19 creating lambda 13A-18 and the EcoRI+BamHI fragment of lambda 13B-96 was further subcloned into the corresponding sites of this construct, creating a cDNA, lambda 13-24, which was determined to be full-length by comparison to published nucleotide sequences and restriction maps (Grundmann et al. 1986; Ichinose et al. 1986a) . The PstI fragment of lambda 13-24 was further subcloned into the expression vector, pPH3, and called pPH4 (ATCC 40778) (Fig. 1). pPH3 (ATCC 40777) is comprised of the EcoRI+BamHI fragment of pBM150 (Johnston and Davis 1984) containing the divergent GAL1-GAL10 mutated promoter of Saccharomvces cerevisiae subcloned into the multiple cloning region of YEp352 (Hill et al. 1986) . Yep352 was designed as a yeast shuttle vector and is, therefore, capable of autonomous replication in both yeast and bacteria. Additionally, this shuttle vector provided the necessary multiple cloning region derived from pUC18 which contained unique restriction sites not present elsewhere in the shuttle vector. Furthermore, the size of this shuttle vector was 5181 base pairs providing the inventor with a minimally-sized source of the necessary yeast replication and selection sequences.

(b) Bacterial and yeast strains and growth conditions

Escherichia coli strain TB-1 was used to propagate all plasmids according to standard methods (Maniatis et al. 1982) . §.. cerevisiae strain DKy461 (a/α ura3-52/ura3- 52 his3-l/his3-l trpl-289/+) was transformed by the lithium acetate protocol (Ito et al. 1983) . Yeast cells were grown in yeast extract-peptone containing glucose (YEPD; Sherman et al. 1983) before transformation a.nd selected for uracil prototrophy on synthetic media plates supplemented with glucose, but lacking uracil (SD-URA; Sherman et al. 1983) after transformation.

(c) Preparation of yeast cell-free protein extracts

Yeast cell-free extracts were prepared by growing transformants in synthetic media supplemented with galactose (1% v/v) , but lacking uracil (SG-URA; Tamaki and Aoki 1981) to an absorbance of 0.2-0.4 at 540 nm. Cells were harvested by centrifugation, resuspended in 16 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM PMSF, 2 μg/ml leupeptin and 0.1% Tween 20) and disrupted in a Biospec Products Bead-beater (Biospec Products, Bartlesville OK) at 2'C (5 x 1 - min cycles with 2 min intervals between each cycle) . The cell extract was centrifuged at 5000 x g for 10 min. The supernatant fraction was applied to a Whatman DE52 (microgranular) column (Whatman Biosystems Ltd. , Kent UK) equilibrated with 50 mM Tris-HCl, pH 7.5, 1 mM EDTA and eluted with 0.2 M NaCl, 1 mM PMSF and 2μ leupeptin/ml. The resulting eluent was then passed over a Sephadex G-25 (Pharmacia, Piscataway NJ) equilibrated with 50 mM Tris-HCl, pH 7.5, 1 mM EDTA buffer. The protein concentrations of these samples were estimated using the Bio-Rad microassay procedure (Bio-Rad, Richmond CA) .

(d) Electrophoresis and Western blot procedures

All samples were subjected to electrophoresis in 7.5% SDS-PA gels (Weber and Osborn 1969) . Electrophoresis was carried out at 1.2 V/cm² at room temperature. Proteins were electrotransferred onto nitrocellulose at 200 milliAmperes overnight. The filter was incubated in TBST containing 3% w/v gelatin and 1% w/v bovine serum albumin for 30 min, washed 3 times in TBST and incubated for 1 h with a rabbit polyclonal anti-factor Xllla antibody (Calbiochem) diluted 1000-fold in TBST. The filter was again washed in TBST, and incubated for 45 min with goat anti-rabbit IgG (BioRad) conjugated with HRP diluted 1000- fold in TBST. The filter was washed in TBS (50 mM

Tris-HCl, pH-8.0, 150 mM NaCl) and finally developed with HRP color reagent (BioRad) .

(e) Factor XIII assays

Factor XIII assays were performed by mixing 45 μl of 20 mg/ml fibrinogen, 100 μl of 10 mM cysteine, 150 mM NaCl, adjusted to pH 7.0 with Tris base, 10 μl of 50 mM CaCl₂, 50 μl of sample and 5 μl of 100 units/ml thrombin. After incubating at room temperature for 3-5 min, the fibrin clots were quantitatively transferred with the use of 10 μl glass capillary tubes into 100 μl of 8.0 M urea, 1% SDS. After the clot became visibly dissociated, 10 μl of these mixtures were subjected to electrophoresis in 7.5% SDS PA gels (Weber and Osborn 1969) and checked for the presence of gamma-chain dimers.

RESULTS AND DISCUSSION

(a) Construction of factor XIII expression vector

In order to express human placental factor XIII in yeast, a full-length placental factor XIII cDNA was cloned. The cDNA was determined to be full-length by comparison to previously published nucleotide sequences and restriction maps (Grundmann et al. 1986; Ichinose et al. 1986a; Takagi and Doolittle 1974) . The cDNA was placed under the control of the GAL1 promoter using a yeast expression vector, pPH3, which contains yeast GAL1- GAL10 promoter (Johnston and Davis 1984). The resultant factor XIII expression plasmid, pPH4 (Fig.l), was transformed into yeast strain Dky 61.

(b) Analysis of yeast cell-free protein extracts for presence of biologically functional factor XIII

Cell-free extracts prepared by mechanical disruption of the yeast cells were analyzed by Western-blotting using a polyclonal anti-factor Xllla antibody. Extracts from pPH4 transformants displayed one distinct band on Western blots (Fig. 2A, lane 5) , while control extracts (Fig. 2A, lane 1) did not display detectable immunoreactive products. The factor XIII displayed a similar mobility (Fig. 2A) in SDS-PA gels compared to placental factor XIII (apparent molecular weight of 81 kDa) . Since thrombin cleaves factor XIII in a specific manner, it was used to further test the protein. The expected thrombin cleavage products of placental factor XIII would include 79-kDa, 56-kDa, and 4-kDa (not detectable on Western blots) fragments. Thrombin cleaved the factor XIII (Fig. 2A, lane 6) into similar size fragments. The fragment of the protein corresponding to the 24-kDa fragment of the placental protein was undetectable on all Western blots. It was discovered subsequently that the 24 kDa fragment, whether it originated from the protein or the placenta1- derived protein, was susceptible to proteases present in the yeast extract.

The extracts that demonstrated immunopositive signals on Western blots also showed factor XIII activity (Fig. 2B, lanes 5 & 6) . Factor XIII activity was assessed by testing these extracts for the formation of gamma-chain dimers of fibrin. The formation of gamma-chain dimers of fibrin can be seen in Fig. 2B, lanes 3, 5 and 6. The immunopositive signal (Fig. 2A, lane 5) and the formation of gamma-chain dimers could only be the result of the expression of factor XIII since the host strain containing only the parent plasmid, pPH3, did not exhibit the same results (Fig. 2B, lane 4) . The amount of factor XIII synthesized by pPH4 transformants constitutes approximately 1-2% of the partially purified cell-free extract protein. The growth medium did not contain any detectable levels of factor XIII.

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REFERENCES CITED

The following references to the exten that they provide procedural details supplementary to those set 5 forth herein, are specifically incorporated herein by reference.

1. Benton and Davis, Science 196:180-182 (1977)

2. Bonn, Mol. Cell. Biochem. 20:67-75 (1978)

10. 3. Broker and Bauml, FEBS Letters 248:105-110 (1989)

4. Chen and Seeburg, DNA 4:165-170 (1985)

5. Chung et al., J. Biol. Chem. 249:940-950 (1974)

6. Egbring, et al.. Blood 49:219-231 (1977)

7. Golf, et al.. Gene 27:35-46 (1984)

15 8. Grundmann, et al., Proc. Natl. Acad. Sci. USA 83:8024-8028 (1986)

9. Henriksson, et al., Scand. J. Haem. 14:355-360 (1975)

10. Hill et al.. Yeast 2:163-167 (1986)

11. Ichinose et al., Biochem. 25:6900-6906 (1986) 0 12. Ichinose et al., Biochem. 25:4633-4638 (1986)

13. Ito, et al., J. Bact. 153:163-168 (1983)

14. Jagadeeswaran and Kaul, Gene Analysis Techniques 3:79-85 (1986)

15. Johnston and Davis, Mol. Cell. Biol. 4:1440-1448 5 (1984)

16. Kimitsuji et al., Eur. J. Pediat. 146:519-523 (1987)

17. Mackay, et al., EP0314096, published May 3, 1989 (based upon U.S. patent application No. 189,547, filed May 3, 1988 and No. 116,095 filed October 29, 0 1987).

18. Maniatis et al., Molecular Cloninσ: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)

19. Miyajima, et al., Nuc. Acids Res. 12:6397-6414 (1984) 5 20. Nilsson et al., Ann. Surσ. 182:677-682 (1975)

21. Nussbaum and Morse, Blood 23:669-678 (1964) 22. Sherman et al., Methods in Yeast Genetics, pp. 61-64, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983)

23. Takagi and Doolittle, Biochem. 13:750-756 (1974) 24. Takahashi et al., Proc. Natl. Acad. Sci. USA 83:8019- 8023 (1986)

25. Tamaki and Aoki, Biochem. Biophys. Acta. 661:280-286 (1981)

26. Weber and Osborn, J. Biol. Chem. 244:4406-4412 (1969) 27. Young and Davis, Proc. Natl. Acad. Sci. USA 80:1194-

1198 (1983)

* * * * * * * *

The present invention has been described in terms of particular embodiments found or proposed to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For instance, proteins other than Factor XIII are anticipated to be used in conjunction with the vector of the invention. More particularly, since there are apparently a family of enzymes which are grouped within the meaning of Factor XIII, all such Factor XIII analogues are expressly anticipated here. All such modifications are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a yeast expression vector encoding a polypeptide, comprising:

(a) obtaining a DNA fragment encoding a polypeptide;

(b) obtaining a fragment of yeast DNA containing a GAL1-GAL10 promoter region;

(c) cleaving a yeast cloning vector containing a multiple cloning region;

(d) inserting the fragment of yeast DNA containing the promoter region into the yeast cloning vector so that a GAL1 promoter is in close proximity to the multiple cloning region; and

(e) inserting the DNA fragment encoding the polypeptide into the multiple cloning region of the vector produced in step (d) so that the GALl promoter is arranged in a transcriptional and translational unit with the polypeptide.

2. The method of claim 1 wherein the DNA fragment encoding the polypeptide is derived from a human placental cDNA library.

3. The method of claim 1 wherein the fragment of yeast DNA containing a GAL1-GAL10 promoter region is derived from pBM150. -27-

4. The method of claim 1 wherein the yeast cloning vector is YEp352.

5. The method of claim l wherein the polypeptide is further defined as a blood coagulation factor.

6. The method of claim 5 wherein the blood coagulation factor is further defined as a Factor XIII polypeptide.

7. The method of claim 6 wherein the Factor XIII is further defined as a Factor Xllla polypeptide.

8. A yeast expression vector comprising a DNA sequence encoding a polypeptide in a transcriptional and translational unit with the GAL1 promoter, the DNA sequence encoding a polypeptide being inserted into a multiple cloning region of the yeast expression vector 3^• of the promoter.

9. The yeast expression vector of claim 8 wherein the polypeptide is further de ined as a blood coagulation factor.

10. The yeast expression vector of claim 9 wherein the blood coagulation factor is further defined as a Factor XIII polypeptide. 14780

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11. The yeas expression vector of claim 8 wherein the polypeptide is further defined as a Factor Xllla polypeptide.

12. The vector of claim 8 defined further as being adapted for expression in an eukaryotic host.

13. A process for producing a substantially purified polypeptide using a yeast host cell which contains the yeast expression vector of claim 8.

14. The process of claim 13 wherein the polypeptide is further defined as a blood coagulation factor.

15. The process of claim 13 wherein the polypeptide is further defined as a Factor XIII polypeptide.

16. The process of claim 13 wherein the polypeptide is further defined as a Factor Xllla polypeptide.

17. A host cell comprising the yeast expression vector of claim 8.

18. The host cell of claim 17 wherein the polypeptide is further defined as a blood coagulation factor.

19. The host cell of claim 17 wherein the polypeptide is further defined as a Factor XIII polypeptide. -29-

20. The host cell of claim 17 wherein the polypeptide is further defined as a Factor Xllla polypeptide.

21. The host cell of claim 16 further defined as being a yeast.

22. The host cell of claim 16 further defined as being a prokaryote.

23. The host cell of claim 21 wherein the yeast is further defined as Saccharomyces.

24. The host cell of claim 22 wherein the prokaryote is further defined as Escherichia coli.

25. The host cell of claim 16 wherein the DNA segment encoding a polypeptide is under transcriptional control of regulatory signals functional in the host cell, said regulatory signals controlling expression of the polypeptide to allow transcriptional and post-transcriptional modification.

26. The host cell of claim 25 wherein the polypeptide is further defined as a blood coagulation factor.

27. The host cell of claim 25 wherein the polypeptide is further defined as a Factor XIII polypeptide.

28. The host cell of claim 27 wherein the polypeptide is further defined as a Factor Xllla polypeptide.

29. A method of producing a polypeptide, comprising:

(a) producing a host cell including a yeast expression vector, said vector comprising a DNA sequence encoding a polypeptide in a transcriptional and translational unit with the

GAL1^" promoter, the DNA sequence being inserted into a multiple cloning region of the yeast expression vector 3' of the promoter, said host cell being capable of expressing the polypeptide;

(b) culturing the host cell under conditions facilitating expression of the polypeptide; and

(c) recovering the polypeptide.

30. The method of claim 29 where the polypeptide is further defined as a blood coagulation factor.

31. The method of claim 29 where the polypeptide is further defined as a Factor XIII polypeptide.

32. The method of 29 where the polypeptide is further defined as a Factor Xllla polypeptide.

33. The method of claim 29 where step (c) further comprises recovering the polypeptide as a percentage of the protein of the partially purified cell-free extract in the host cell of about 2%.

34. A method of producing a Factor Xllla polypeptide, comprising:

(a) producing a yeast host cell including a yeast expression vector, said vector comprising a DNA sequence encoding a Factor Xllla polypeptide in a transcriptional and translational unit with the GAL1 promoter, the DNA sequence being inserted into a multiple cloning region of the yeast expression vector 3' of the promoter, said host cell being capable of expressing the Factor

Xllla polypeptide;

(b) culturing the host cell under conditions facilitating expression of the Factor Xllla polypeptide; and

(c) recovering the Factor Xllla polypeptide.

35. A vector having the characteristics of that vector deposited with the American Type Culture Collection as ATCC number 40777.

36. A vector having the characteristics of that vector deposited with the American Type Culture Collection as ATCC number 4077.