EP0156851A1 - Yeast hybrid vectors - Google Patents

Abstract

A yeast vector comprises one or the other or the two REP1 and REP2 genes derived from the yeast plasmid 2mu, located with respect to a regulatory sequence capable of ensuring the expression of one or of the two REP1 genes , REP2 where the regulatory sequence comprises an activator which acts substantially independently of the number of reproductions of the vector in a host organism transformed with the vector. The regulatory sequence includes a controllable activator such as the yeast activator PGK which allows control of the number of reproductions of expression vectors transformed in common with the yeast vector. The yeast vector, which can be integrated into the chromosomal genome of yeast, is used in a method of producing a polypeptide in which the production of the polypeptide is kept at a low level until reaching a predetermined culture cell density; at this point, the number of reproductions of the expression vector and / or the level of expression is increased by modifying the culture conditions.

Description

Yeast hybrid vectors

Field of the Invention

This invention relates to the field of recombinant DNA biotechnology. In particular it relates to a yeast vector, a yeast host organism and a process for the production of a polypeptide.

Background to the. Invention

There are now numerous examples of polypeptides which may be produced using the techniques of recombinant DNA biotechnology. In broad terms, these techniques involve the insertion of a gene coding for a desired polypeptide into a vector capable of stable existence in a host organism. The gene is inserted into the vector in a position relative to appropriate control sequences, such that once within a host organism, the vector expresses the inserted gene to produce the polypeptide. Such vectors are referred to in the art as "expression vectors" and have been the subject of considerable research in recent years. The main thrust of the research has been to develop expression vectors which are compatible with the well characterised host organisms (i.e. bacteria, yeasts and mammalian cells) and which produce high yields of polypeptide. This has involved detailed study of the control sequences affecting expression and, in particular, promoter sequences. Many promoters have been identified and used to produce efficient expression vectors.

The amount of protein produced by a transformed host organism is however limited by the number of vectors stably existing within the cells of the organism (hereinafter referred to as the vector copy number). The object of the present invention is to provide a yeast vector capable of increasing the copy number of a yeast expression vector in a yeast host organism.

We have recently described in our copending published European patent application EP-A2-0073635, a set of plasmid vectors which are useful for the expression of polypeptides in yeast (see also Tuite et al EMBO J. (1982)1 603; Dobson et al NAR (1983) 11 2287; Mellor et al Gene (1983) 24 , 1 - 14). One of the plasmids described makes use of the control sequences of the yeast phosphoglycerate kinase (PGK) gene and the yeast 2μ origin of replication. This plasmid, and indeed other yeast plasmids based on the yeast wild type 2μ plasmid, all require the presence of endogenous 2μ plasmids to provide functions in trans that permit efficient replication amd maintenance of high copy number. These functions are encoded by the REP1 and REP2 genes (Broach, Cell (1982) 28 203). 2μ plasmid replication usually occurs once during the early S phase (Zokian et al Cell (1978) 17, 923). However, when the copy number is low, plasmid replication is uncoupled from the cell cycle and replication occurs until the normal copy number is again established (see Broach loc cit). The products of the REP1 and REP2 genes are responsible for enhanced replication in normal circumstances under conditions of low copy number.

Summary of the Invention

According to a first aspect of the present invention we provide a yeast vector including either or both of the REP1 and/or REP2 gene(s) derived from the yeast 2μ plasmid, located relative to one or more control sequences directing expression of the REP1 and/or REP2 gene(s), characterised in that at least one of the control sequences includes a promoter which operates substantially, independently of copy number in a host organism transformed with the vector.

The vector of the present invention allows for the production of the REP1 and/or REP2 gene products in a host organism, independent of the usual control applied to the genes in their natural position in the yeast wild type 2μ plasmid. The production of the gene product or products is controlled by the control sequences in the vector and is independent of copy number. This allows for the enhancement of copy number in yeast expression systems providing a high yield of desired polypeptide. The yeast vector of the present invention may, for example, be used to cotransform a yeast host organism with a 2μ-derived yeast expression vector capable of expression of the desired polypeptide. The yeast vector of the present invention is capable of increasing the copy number of the cotransforming expression vector and hence increasing yield.

The commercial use of expression systems is hampered by the lethal or debilitating effect of toxic expression products upon the host organism. In the expression vectors described in our copending published European patent application EP-A2-0073635, the PGK control sequences include a promoter which is capable of regulation by the level of fermentable carbon in the culture medium of the host organism. It is possible, using this system, to regulate gene expression to produce a 30-fold difference in product level between a glucose medium and an acetate medium. Other yeast expression vectors have been described which afford expression level control through the use of promoters which respond to other external influences, (see, for example, published British patent application GB2104902A).

In a controlled expression system, the host organism can be cultured to produce a high cell density whilst expression of a gene in an inserted vector is kept to a minimum. When the host organism reaches an appropriate cell density, expression may be induced, for example, by the addition of material to the culture medium, thereby causing abundant product formation. The viable cell density will be reduced by the production of polypeptides which are lethal to the host organism but the yield of product is maintained for a short time at a high level.

The known controlled expression systems involving yeasts do not have the ability to control expression over a wide range of expression levels. In the case of the system described in copending European patent application EP-A2-0073635 and by Tuite et al (loc cit) it is not possible to reduce the concentration of toxic products to the point where they have no effect upon cell growth.

Preferably, the yeast vector of the invention includes a promoter which directs expression of either or both of the REP1 and/or REP2 gene(s) and is controllable by an external agent. The external agent may be any alteration of conditions applied to the vector (for example, a change of temperature) but is preferably in the form of a material added to the yeast host organism culture medium.

A yeast vector of this preferred type allows for the control of vector copy number. The yeast vector may be used to cotransform a yeast host organism, and heterologous polypeptide production may be controlled, not only by control of the promoter directing polypeptide expression, but also by controlling the copy number of the expression vector using a yeast vector of the present invention.

Preferably the promoter included in the yeast vector of the present invention is derived from the yeast PGK gene and the external agent is a source of fermentable carbon, such as glucose.

The yeast vector may include one of the REP1 or REP2 genes or both genes.

Preferably the yeast vector includes a DNA sequence capable of causing integration of the yeast vector with the chromosomal genome of a yeast host organism. The vector may, for example, include a portion of the yeast HIS3 gene and a portion of phage λ DNA.

In a seeond aspect of the invention we provide a yeast host organism transformed with a yeast vector according to the first aspect of the invention. The yeast vector may be maintained episomally or may be integrated into the yeast chromosome. The yeast host organism may be transformed with one yeast vector of the invention, including either or both of the REP1 and/or REP2 genes, or with two vectors, one including the REP1 gene and the other including the REP2 gene. The yeast host organism may be a diploid strain (such as yeast strains MDX1 or MDX2 described herein) or a haploid strain (such as MDX3 described herein). The host organism may additionally be transformed with a yeast expression vector having an origin of replication derived from the 2μ yeast plasmid and including a gene coding for a heterologous polypeptide. The copy number of such an expression vector may be enhanced and/or controlled by the yeast vector of the invention. The term "heterologous" as used herein denotes a polypeptide not naturally occurring in yeast. The term "polypeptide" as used herein denotes any polypeptide and includes proteins. Examples, of suitable polypeptides inelude hormones (such as growth hormones) and enzymes (such as chymosin). The gene coding for the heterologous polypeptide is located in the yeast expression vector relative to control signals capable of directing expression of the polypeptide. The control signals may include the same or a different promoter, to the promoter included in the yeast vector of the invention. Preferably the yeast is Saccharomyces cerevisiae. In a third aspect of the invention we provide a process for the production of a polypeptide comprising the steps of culturing a yeast host organism cotrans-formed with a vector of the present invention and a yeast expression vector including a gene coding for the polypeptide and having an origin of replication derived from the yeast 2μ plasmid.

Preferably the yeast host organism is cultured under conditions in which production of the polypeptide is at a first level for a period of time to produce a predetermined cell density at which density the conditions are changed to cause polypeptide production at a second, higher level. The production level may be controlled by independent or simultaneous control of expression and/or vector copy number.

Preferably the yeast host organism is cultured under conditions in which the copy number of the expression vector is at a first level for a period of time, to produce a predetermined culture cell density at which density the conditions are changed to give a copy number of the expression vector at a second level.

Most preferably the conditions are changed to cause a simultaneous expression level and copy number increase at the predetermined cell density.

Brief Description of the Drawings

The invention is illustrated by the following description of embodiments of the invention with reference to the accompanying drawings, in which:-

Figure 1 - shows a restriction site map of plasmid pJDB219 (I.R. = inverted repeat),

Figure 2 - shows a restriction site map of plasmid pMA500, Figure 3 - shows a restriction site map of plasmid pMA 301, Figure 4 - shows a restriction site map of plasmid pMA 401, Figure 5 - shows a restriction site map of plasmid

YRp7, Figure 6 - shows a restriction site map of plasmid pMA 402, Figure 7 - shows a restriction site map of pMA403 (pMA404),

Figure 8 - shows a restriction site map of plasmid pMA 405, Figure 9 - shows the structure of plasmid pMA505, Figure 10- shows the recombination events involved in transformation and integration.

In the drawings the following abbreviations are used:

R = EcoRI B = BamHI

Xb = Xbal Bg = Bg1ll P = Pstl C = Cla I Pv = PvuII H = Hindlll

Description of Embodiments

The generial methods used are as described in published European patent application EP-A2-0076335, the contents of which are incorporoated herein by reference. 1. Subcloning of the REP1 and REP2 genes

The entire nucleotide sequence of the 2μ plasmid has been determined by Hartley and Donelson (Nature (1980) 286, 860). The coordinates used in Figures 1 and 2 are B-form coordinates .

In order to prepare REP1 and REP2 for expression from the PGK promoter the following manipulations were carried out:

Plasmid pJDB219 which contains the REP1 and REP2 genes (Beggs (1978) Nature 275, 104) (Figure 1)) was cut with EcoRI and PvuII and then ligated, at low ligase concentration, with pBR322, cut with EcoRI and BamHI to join the cohesive EcoRI ends. BamHI linkers (CCGGATCCGG) were added and ligated at high ligase concentration to add BamHI linkers to the PvuII blunt ends. This mixture was then treated with BamHI and religated to join BamHI ends. The resulting products were used to transform E.coli strain AKEC28 to ampicillin resistance and the resulting colonies were screened to find a plasmid with the structure shown in Figure 2. This plasmid was designated pMA500. Plasmid pMA 500 was cleaved with Xbal and treated with nuclease Bal 31 to remove 150-360bp. This deleted pool was ligated in the presence of an excess of BamHI linker (CCGGATCCGG). Individual deleted derivatives were analysed by sequence analysis to find derivatives with new

BamHI sites within 100bp of the termination codons of REP1 and REP2. Such a derivative with a BamHI site within 100bp of the REP1 termination codon was designated pMA501 and a similar derivative with the new BamHI site within 100bp of the REP2 termination codon was designated pMA502.

2. Construction of a Yeast Vector including the REP1 gene

The BamHI fragment containing the REP1 coding sequence from pMA 501 was inserted, in the correct orientation, into the Bglll expression site of plasmid pMA3013 (published Euroean patent application EP-A2-0073635). The resulting plasmid, designated pMA3013-REPl was then used to transform yeast strain MD40-4C with and without an endogenous 2μ plasmid. The frequency of transformation, the stability of the transformants and the copy number of the plasmid was compared with data obtained with the expression vector alone (Table1). Both plasmids transform a 2μ⁺ host at about the same frequency and both are stable, but pMA3013-REP1 has a somewhat lower copy number than pMA3013. As expected pMA3013 transforms a 2μ° (lacking endogenous 2μ plasmid) at a very low frequency. Further analysis has shown that all transformants arising from pMA 3013 are due to recombinational exchanges across the chromosomal LEU2 region, i.e. there is no plasmid present in these transformants. This is because the REP1 and REP2 functions needed for 2μ- derived plasmid replication are lacking in this strain. In contrast, plasmid pMA3013- REP1 transforms at a high frequency and its copy number per plasmid bearing cell is very high. (In fact it exhibits the highest reported copy number of any 2μ-based plasmid). This shows that REP1 is being expressed independently of copy number control and that its over-production leads to very high copy number. The gene is expressed from the PGK promoter which has been shown elsewhere (see published European patent application EP-A2-0073635) to. be controllable by adjustment of the level of fermentable carbon in the yeast culture medium.

3. Construction of a Yeast Vector including the REP2 gene

The EcoRI-BamHI fragment bearing REP2 from pMA502 was subcloned into pMA1 (Mellor et al) (op cit) partially cleaved with EcoRI and cleaved with BamHI to give pMA503. This vector was then cleaved with Xbal and treated with exonuclease Ba131 to remove 1100bp. This deleted pool was then ligated in the presence of BamHI linkers. Deleted pMA503 derivatives were screened to find one with a new BamHI site within 20bp of the initiating ATG of REP2. Such a derivative was designated pMA504. Plasmid pMA504 contains a BamHI fragment bearing the REP2 gene such that insertion of this BamHI fragment into plasmid pMA3013 (published European patent application EP-A2-0073635) results in PGK promoter-directed expression of REP2, as described in 2 above for the BamHI fragment containing the REP1 gene.

4. Construction of an Integrative Expression Vector

The insertion of REP1 and REP2 into a molecule such as pMA3013 affords PGK control of REP functions. However the presence of these plasmids in a cell together with a plasmid expressing a heterologous gene may lead to reduced copy number of that plasmid due to competition for replication machinery. Also recombination between the different plasmids may lead to undesirable and unpredictable derivatives. To overcome these problems REPl and REP2 are advantageously expressed from the PGK control signals integrated into specific sites in the yeast chromosomal genome. To achieve this a generally useful integrative expression vector is constructed.

The Clal-Pstl fragment containing the 5' region of

PGK from the Bg1ll expression site to -820 from pMA301 (Figure 3) was used to replace the large Pstl-Clal fragment of pBR322 to yield plasmid pKA401 (Figure 4). Plasmid pMA401 was then cleaved with EcoRI and Pstl and ligated with an EcoRI-Pstl digest of YRp7 (Figure 5). After transformation of AKEC28 to tryptophan independence, colonies were screened for the presence of a plasmid of the structure shown in Figure 6. This plasmid is designated pMA402. The large Bglll-Pstl fragment of pMA402 is then replaced with the large Bg1ll-Pstl fragment of pMA3013 (published European patent application EP-A2- 0073635) to produce pMA403 (Figure 7). Plasmid pMA403 is then cleaved at the unique Pstl site, blunt ended with SI nuclease and then ligated in the presence of BamHI linkers (CCGGATCCGG). After transformation of AKEC28 colonies are screened for a derivative of pMA403 which has its Pstl site replaced by a BamHI site. Such a derivative is pMA404 (Figure 7). Plasmid pMA405 is a pBR322 derivative containing a 1.8kb BamHI fragment designated Sc2715 (Struhl and Davis 1980 J. Mol. Biol. 136, 309) which contains the yeast HIS3 region and 242bp of phage, λ DNA at one end (Figure 8). This molecule is partially cleaved with Bglll and ligated with BamHI digested pMA404. After transformation of AKEC28 colonies were screened for plasmids of the structure shown in Figure 9. Such a plasmid is pMA505.

Plasmid pMA505 is a universal yeast integrative expression vector. Any coding sequence may be introduced into the Bglll expression site and so come under the control of the PGK expression signals. Then the entire BamHI fragment containing the λ sequence, the bisected HIS3 region, the pBR322 sequence from the Hindlll site to the BamHI site, the PGK 3' region, the inserted coding sequence, the PGK 5' region and the TRP1 gene is then cleaved from the pMA505 derivative, purified from an agarose gel and used to transform yeast strain MD40-4C (published European patent application EP-A2-0073635) to tryptophan independence. The HIS3 homologous end will direct integration to the HIS3 locus and the λ homologous end ensures that the recombination event leads to replacement rather than duplication. The integrated fragment is therefore relatively stable (Roeder and Fink, 1982 PNAS 79 , 5621 ) . 5. Expression and Integration of the REP1 and REP2 genes

The BamHI fragments containing the REP1 and REP2 fragments from pMA503 and 504 are inserted into the Bg1ll expression site of pMA505 to generate pMA506 (REP1)and pMA507 (REP2) respectively. The resulting BamHI fragments from pMA506 and 507 are then used to transform yeast strain MD40-4C cir° (α, ura2, trp1, leu2-3, leu2-112, his3-11, his3-15), in the case of the REP1 fragment, and MDx.cirº (a, leu 2-3 leu2-112 trp1, his4-519, in the case of the REP2 fragment, to tryptophan independence. The cir° derivatives are constructed according to Dobson et al (1980 Curr. Genet. 2, 201). The recombination events involved in the transformation and integration are shown in Figure 10. This results in two haploid strains designated MDX1 and MDX2 which carry REP1 and REP2 respectively, integrated at their HIS3 loci and expressed under the control of PGK. These two strains are crossed to produce the diploid MDX3 which therefore contains both REP gene expression configurations as alleles of the HIS3 locus. Plasmids such as pMA3013 can be used to transform this diploid and the resulting transformants then have expression vector copy number controlled via PGK promoter control of the integrated copies of REP1 and REP2.

Claims

CLAIMS :

1. A.yeast vector including either or both of the

REPl and/or REP2 gene(s) derived from the yeast 2μ plasmid, located relative to one or more control sequences directing expression of the REPl and/or REP 2 gene(s), characterised in that at least one of the control sequences includes a promoter which operates substantially independently of vector copy number in a host organism transformed with the vector.

2. A yeast vector according to claim 1 wherein the promoter which directs expression of either the REP1 and/ or REP2 gene(s) is controllable by an external agent.

3. A yeast vector according to claim 2 wherein the promoter is derived from the yeast phosphoglyceratekinase gene and the external agent is a source of fermentable carbon.

4. A yeast vector according to claim 1 wherein the vector includes a DNA sequence capable of causing integration of the yeast vector with the chromosomal genome of a yeast host organism.

5. A host organism transformed with a yeast vector according to claim 1.

6. A host organism according to claim 5 transformed with a yeast expression vector having an origin or replication derived from the 2μ yeast plasmid and including a gene coding for a heterologous polypeptide.

7. A process for the production of a polypeptide comprising the steps of culturing a yeast host organism cotransformed with a vector according to claim 1 and a yeast expression vector having an origin of replication derived from the 2μ yeast plasmid and including a gene coding for a heterologous polypeptide.

8. A process according to claim 7 wherein the yeast host organism is cultured under conditions in which pro duction of the polypeptide is at a first level for a period of time, to produce a predetermined culture cell density at which density the conditions are changed to cause polypeptide production at a second, higher level.

9. A process according to claim 8 wherein the yeast host organism is cultured under conditions in which the copy number of the yeast expression vector is at a first level for a period of time, to produce a predetermined culture cell density, at which density. the conditions are changed to cause an increase in copy number of the yeast expression vector to a second, higher level.