EXPRESSION VECTOR

Abstract: A yeast expression vector comprising a promoter and a downstream activator sequence wherein the downstream activator sequence is positioned relative to the promoter to enhance promoter activity and is positioned in the vector such that translation of the downstream activator sequence does not occur. The yeast phosphoglycerate kinase (PGK) downstream activator sequence is exemplified together with a synthetic oligonucleotide downstream activator sequence having the nucleotide sequence: ACCAAACGGTGAAAGAAACA. The promoter and downstream activator sequence may be homologous (e.g. PGK promoter/PGK downstream activator sequence) or heterologous (e.g. TRP promoter/PGK downstream activator sequence).

Description: EXPRESSION VECTOR Field of the invention This invention relates to recombinant DNA biotechnology and in particular to a yeast expression vector Background to the invention There are now a wide variety of vectors available for the expression of heterologous polypeptides in various host organisms. Heterologous polypeptides have been successfully produced, for example in bacteria, yeasts and mammalian cells. The commercial success of processes for preparing polypeptide products by fermentation relies howeve upon the amount and rate of production of the desired polypeptide by a given cell-line. Despite extensive research activity in the area of gene expression, there remains a continued need for vectors capable of expressing heterologous polypeptides at high levels in host organisms. It is known, in connection with mammalian gene expression, that coding sequences within certain mammalian structural genes have an enhancing effect upon the promoter for those genes (see for example osbourne, T.F. e_t a_l_ Mol. and Cellular Biol. (1984), 1293-1305, Wright, S. et al Cell (1984) 3_ , 265-273 and Chamay, p. et al Cell (1984) _3£, 251-263). We have now surprisingly discovered that yeast structural genes also possess downstream activator sequences. Further, we have discovered that translation of the downstream activator sequences is not necessary for enhancement of promoter activity. The term "downstream activator sequence" as used herein denotes a nucleotide sequence within the gene coding for a particular polypeptide which functions to enhance the expression of a homologous gene by the relevant promoter. I however, the promoter is used to express an heterologous gene, the promoter loses activity, apparently due to the absence of the downstream activator sequence. In the field of expression vectors it is known to produce desirable polypeptides by producing them as fusion proteins with an N-terminal portion of a highly expressed gene. such an N-terminal portion may include a downstream activator sequence. It is however, in a number of circumstances, undesirable to produce fusion proteins. They require cleavage to liberate the desired polypeptide, necessarily involve polypeptide production by the host cell over and above production of the desired polypeptide, and may lead to purification difficulties. Brief Description of the Invention According to the present invention, we provide a yeast expression vector comprising a promoter and a downstream activator sequence wherein the downstream activator sequence is positioned relative to the promoter to enhance promoter activity and is positioned in the vector such that translation of the downstream activator sequence does not occur. The vector of the invention includes the downstream activator sequence in such a position that it will enhance promoter activity but is not itself translated into polypeptide. Thus the advantage obtained by expressing genes to produce fusion proteins is obtained without the need for expression of host protein. The downstream activator sequence, may for example, be located relative to a transcription start site such that transcription of the down¬ stream activator sequence occurs, but not translation. In the alternative, the downstream activator sequence may be located in the plasmid such that neither transcription nor translation occurs. The vector may include more than one downstream activator sequence. Multiple copies of the downstream activator sequence may lead to higher expression. The downstream activator sequence may be derived from the structural gene of a yeast polypeptide or may be synthetic. In the preferred embodiment described below, the downstream activator sequence is isolated from t coding sequence of the yeast phosphoglycerate kinase gene (PGK). The promoter and the downstream activator sequence may be heterologous. That is, they may be from different yeast structural gene expression systems. For example, th promoter may be a TRP promoter and the downstream activator sequence may be the PGK downstream activator sequence. Preferably however, the promoter is from a yeast structural gene expression control region and the downstrea activator sequence is from the said structural gene. For example, the promoter may be the PGK promoter and the downstream activator sequence may be the PGK downstream activator sequence. In a second aspect of the invention, we provide th yeast, phosphoglycerate kinase (PGK) downstream activator sequence or a functional derivative or analogue thereof. Linkers may be provided for the ready insertion of the downstream activator sequence in an expression vector. Th sequence may be isolated on an approximately 140 bp Sau3a-BamHI fragment of the PGK coding sequence (see Figure 4). This corresponds to the nucleotide sequence from approximately nucleotide +100 to approximately nucleotide +235 in the coding sequence (135 bp without the restriction sites). The active part of the sequence appears further t be located within the part of the fragment between the Accl restriction site and the Ba HI restriction site (see Figure 4). In a third aspect of the invention, we provide a downstream activator sequence having substantially the nucleotide sequence ACCAAACGGTGAAAGAAACA or a functional derivative or analogue thereof. The downstream activator sequence of the third aspect of the invention, which may be synthesised or isolated from geneti material, may be provided at each end with linkers to allo ready insertion into a host vector. suitable such linkers include BamHI and Bglll linkers. The downstream activator sequence shows considerabl homology with the sequences of the yeast Ty element and wit the DEX gene of saccharomyces diastaticus, indicating that downstream activator sequences from these genes may be usef in the invention. The expression vector of the invention may contain relevant control signals and other regulatory regions for t replication of the vector in a host cell, preferably a yeas host cell. The vector, which may be an episomal or an integrating vector, may also comprise one or more selection markers and a restriction site, preferably unique, for the insertion of a gene coding for a heterologous polypeptide. The vector may include a gene coding for a heterologous polypeptide such as a mammalian enzyme (for example, chymosin), hormone (for example, growth hormone) or lymphokine (for example, interferon- 1 ^) . in other aspects of the invention there are provid a yeast transformed with a vector according to the inventio and a process for preparing a polypeptide comprising expressing a gene coding for the polypeptide in an expressio vector according to the invention. Brief Description of the Drawings The invention is now illustrated by the following description and by the accompanying drawings in which Figure 1 shows schematically the functional parts and restriction sites of the various fragments of plasmid DNA, Figure 2 shows schematically the functional parts and restriction sites of i) various fragments of plas DNA (a) and (b) and ii) of plasmid pSK50, Figure 3 shows schematically the functional parts and restriction sites of a fragment of each of plasmi pSK52 and pSK53, Figure 4 shows the N-terminal region of the yeast PGK gene coding region with a sau3a-BamHI fragment includi the downstream activator sequence double underlin Figure 5 shows a deletion series through the yeast TRP1 promoter, Figure 6 shows Northern blots indicating promoter activati by a PGK downstream activator sequence, and Figure 7 shows constructs (Figure 7(a)) including a synthe oligonucleotide downstream activator sequence and Northern blots indicating promoter activation thereby (Figure 7(b)) Detailed Description of the Embodiments 1) Introduction In our copending published European patent application EP-A2-0073635, we describe inter alia the use o the promoter region of the yeast PGK gene in a yeast expression vector. The published patent application describes a number of plasmids using different parts of the 5 f region of the PGK gene, derived by forming a series of deletions of the PGK gene starting at the Sail restriction site closest to the 5' end of the PGK coding sequence and ending around the initiation codon. This deletion series, known as the pMA22a deletion series, gave rise to a range o expression vectors, such as pMA220, pMA230 (includes less nucleotides of the coding sequence) and pMA301 (including n coding sequence nucleotides). We have discovered that the level of heterologous gene expression is higher for those vectors containing at least 230 nucleotides of the PGK codi sequence (e.g. pMA220) than for those vectors including les of the PGK coding sequence. This observed enhancement of promoter function has now been shown to be due to an effect on PGK promoter function rather than on mRNA stability, due to the presence of a downstream activator sequence in the first 230 nucleotides of the PGK coding sequence. A probl arises in the use of vectors such as pMA220 to express heterologous genes, in that they produce unnatural fusion proteins. In the following we describe the location of th PGK downstream activator sequence, its isolation from pMA22 and its reintroduction into another member of the pMA22a deletion series containing one of the PGK coding sequence (pMA301) such that the downstream activator sequence is either contained within the vector transcript but not withi the translation product or is contained neither within transcript, nor translation products. Surprisingly, both these types of constructs allow high level expression of heterologous genes from the PGK promoter. The PGK gene has been fully characterised (Dobson al NAR (1982) _10_, 2625, Perkins et al Biochem. J. (1983) 21 199, Watson e_t al EMBO (1982) _1, 1635). The gene transcri comprises a 40 nucleotide 5' untranslated region, 1245 nucleotide coding region and an 87 nucleotide 3' translated region. The promoter is contained in a 1.5 kb region upstream and adjacent to the transcript. A variety of vectors formed from the pMA22a deletion series are describe in published European patent application EP-A2-0073635. Those referred to herein are pMA220 (see also Tuite e_t al EMBO (1982) _1, 603) pMA230, pMA301 (see also Mellor et al Gene (1985) 3_3, 215) and pMA3-PGK (Dobson et a_l NAR (1982) ^LfJ, 2625 and renamed as pMA27 in Mellor e_t a_l Gene (1983) 7 1). When the entire PGK gene is expressed on a multi-copy plasmid such as pMA27, PGK is produced as 50 to 80% of total cell protein (Mellor et al Gene (1985), 33, 215-226). However, when the PGK coding sequence is replac by a heterologous coding sequence such as, for example, interferon- in plasmids pMA230 and pMA301 to form plasmids PMA230-1 and pMA 301-1 respectively, the yield of heterologous polypeptide is only 1% to 5% of total cell protein. The constructs pMA27, pMA301-l and pMA230-l and their respective RNA levels are shown in Figures la), lb) a lc) respectively. We have now shown that the major reason for these low yields of heterologous proteins is a low level of heterologous mRNA (Mellor ejt al (1985) 3_3_, 215-116). The PGK promoter can therefore direct higher levels of a homologous transcript than of a heterologous transcript and our data suggest that some feature of the PGK coding sequen itself is required to ensure high levels of mRNA. This id has been confirmed by making the plasmid pMA360 (Figure Id) in this construction, an interferon coding sequence is plac approximately 10 bp downstream from the PGK coding sequence This produced a hybrid transcript containing PGK and interferon coding sequences which is translated to produce only PGK. The levels of the transcript per plasmid are as high as those directed by pMA27 and therefore interferon is not simply having a negative effect. Our conclusion is, that by including the whole of the PGK coding sequence with the heterologous transcript, the levels of mRNA are increased. in order to define the precise region of the PGK coding sequence that is required to ensure high mRNA levels we examined some of the promoter fragments from the pMA22a deletion series (see published European patent application EP-A2-0073635) . One of these deletions, namely pMA220, ha an end point at nucleotide 228 within the coding sequence. When interferon is inserted at the BamHI site of pMA220 to produce pMA220-l, (see Figure le)) the observed interferon mRNA levels per plasmid are 5 to 10 fold higher than for vectors such as pMa301-l and pMA230-l. This indicates that the first 228 bp of the PGK coding region is sufficient to produce high levels of heterologous RNA. The most significant region lies between the end points in pMA213 and pMA220 (see Figure 4 which shows the end points of these plasmids) . The use of these vectors to express heterologou genes results in the production of PGK fusion proteins which in many cases is undesirable yet the important region is contained within a coding sequence. We have taken the important region of the PGK coding sequence, the downstream activator sequence, and placed it elsewhere on the vector so that it still functions to enhance the PGK promoter activity but does not necessitate the production of a fusion protein. The downstream activator sequence can be isolated on a 140 b Sau3a-BamHI fragment from pMA220 (double underlined in Figur 4). The following expression vectors containing the downstream activator sequence have been constructed, using techniques and reagents as described in published European patent application EP-A2-0073635. 2. Examples a) Plasmids which contain the downstream activator sequence within the transcriptional unit A plasmid was constructed which contains the 140 bp Sau3a-BamHI fragment referred to above inserted at the Bglll site of pMA301. This plasmid, denoted pMA951 is shown in Figure 2a). An interferon cDNA fragment is inserted downstream of this followed by the PGK terminator fragment. in this case, the downstream activator sequence is contained within the 5' untranslated region of the transcript. Figur 2c) shows a generalised version of this vector for expressin any heterologous coding sequence, denoted pSK50. A plasmid was constructed which contains the downstream activator sequence in the 3' untranslated region of the PGK transcript and which terminates downstream of th downstream activator sequence, in the PGK 3 1 untranslated region. This plasmid, denoted pMA952, is shown in Figure 2b). The promoter is derived from pMA301 and an interfero coding sequence is inserted between the promoter and the downstream activator sequence. b) Plasmids which contain the downstream activator sequence within the PGK promoter but outside the transcriptional unit A plasmid was constructed which contains the downstream activator sequence inserted between the PGK upstream activator sequence and the mRNA initiation point. ' This plasmid, denoted pSK52 is shown in Figure 3a). The site of insertion can be varied according to the position o a synthetic Bglll site which has been inserted at multiple locations throughout the promoter region. A plasmid was constructed which contains the downstream activator sequence upstream of the PGK upstream activator sequence. This plasmid, denoted pSK53 is shown Figure 3b) . The site of insertion of the downstream activator sequence can be varied by placing synthetic Bglll sites at various points in the promoter region. The plasmids pMA951, pMA952, pSK50 pSK52 and pSK53 are all improvements over the original pMA22a deletion seri because they contain a downstream activator sequence which enhances transcription but does not have to be included within the translation product. c) Activation of the TRPl promoter by the 140 bp downstream activator fragment A deletion series through the TRPl promoter linked at the ATG to the human interferon< -2 gene was prepared using standard techniques (see Figure 5). The full promot (fragment 10) produces two transcripts, I, which starts around -190 and II, which starts around -40. The downstre activator sequence fragment was inserted (Bglll-BamHI fragment - the 140 bp fragment was cloned into an SP vector at the BamHI site and excised as a BamHI-Bglll fragment, th Bglll site being at the Sau3a or 5' end of the fragment) in the deletion end points, in the orientation found normally PGK and the RNA levels were quantitated by Northern blottin (see Figure 6(a) and 6(b). The downstream activator sequence shows clear activation in deletions 14 and 23 (see Figure 6(a)) of the small.transcript, in each track the numerals indicate the fragments of the deletion and the superscript "+" indicates the presence of the downstream activator sequence. The upper bands in each track arise from read through transcription. (Figure 6(b) shows a PGK loading control. Plasmid copy number is the same in all constructions. Figure 6(b) also shows another type of activation by the downstream activator sequence. In this case, the downstream activator sequence is located within t transcriptional unit and it is activating. Transcription (as shown by strand specific probes) is in the direction aw from TRPl. d) Downstream Activator Sequence Oligonucleotide Deletion analysis together with RNA gel retardatio studies have suggested that the functional region of the downstream activator sequence is located in the PGK downstream activator fragment between the Accl site and the end of the fragment at nucleotide +235. An oligonucleotid was constructed, using standard synthetic techniques, for part of this region which shows homology with activator sequences in the yeast Ty element and with sequences in the DEX gene of s. diastaticus. The DEX gene exhibits two regions of homology, these being the core sequence GGTGAAAC and TGGTGAAAT at positions 336 and 516 respectively (countin from the "A" of the ATG translation initiation codon) . The Ty element exhibits at. least two regions of homology, these being the sequences TACTTTCCACCT and GACTTTCCAAA in the lowe DNA strand at positions 361-371 and 540-550 respectively (counting from the ATG codon) .The oligonucleotide has the sequence ACCAAACGGTGAAAGAAACA to which linkers were added. The downstream activator sequence oligonucleotide was placed at the Bglll expression site in pMA91 to regenerate a Bglll site for insertion of th interferon gene. The resulting construct is designated pMA960-l (see Figure 7(a)). A control construct without th downstream activator sequence is designated pMA940-l (see Figure 7(a)). in Figure 7, the interferon region is shaded and the downstream activator sequence is denoted by the blac rectangle. Bg = Bglll. The fragments were inserted into the 2JU based plasmid pMA91. A five-fold activation in transcription was shown by Northern blot analysis of total yeast RNA hybridised with a HindiII PGK fragment (Figure 7(a)) and a BamHI interferon fragment (Figure 7(b)). A cop number control experiment confirmed that the copy numbers were compatible in each case. It will be understood that the invention is described by way of example only and modifications of detail may be made within the scope of the invention.

Claims: CLAIMS 1. A yeast expression vector comprising a promoter and a downstream activator sequence wherein the downstream activator sequence is positioned relative to the promoter to enhance promoter activity and is positioned in the vector such that translation of the downstream activator sequence does not occur. 2. A yeast expression vector according to claim 1 wherein the promoter is derived from a yeast structural gene expression control region and the downstream activator sequence is derived from said structural gene. 3. A yeast expression vector according to claim 2 wherein the promoter is the yeast PGK promoter and the downstream activator sequence is a yeast PGK downstream activator sequence. 4. A * yeast expression vector according to claim 1 wherein the promoter and the downstream activator sequences are derived from different yeast structural gene expression systems. 5. A yeast expression vector according to claim 4 wherein the promoter is a yeast TRP promoter and the downstream activator sequence is a yeast PGK downstream activator sequence. 6. A yeast PGK downstream activator sequence, or a functional derivative or analogue thereof. 7. A downstream activator sequence having substantiall the nucleotide sequence: ACCAAACGGTGAAAGAAACA or a functional derivative or analogue thereof, optionally provided at each- end with linkers. 8. A yeast transformed with an expression vector according to any of claims 1 to 5. 9. A process for the production of a polypeptide comprising expressing a gene coding for the polypeptide in expression vector according to any one of claims 1 to 5.

Inventors: KINGSMAN ALAN JOHN, KINGSMAN SUSAN MARY, KINGSMAN, ALAN, JOHN, KINGSMAN, SUSAN, MARY
Applicants: KINGSMAN ALAN JOHN, KINGSMAN SUSAN MARY, KINGSMAN, ALAN, JOHN, KINGSMAN, SUSAN, MARY
Application year: 1986
Main classification: C12N15
IPC codes: C07K14, C12N15