DK173763B1 - Vector for expression in yeast with a PyK promoter, and a process for synthesizing hepatitis B virus surface antigen with the aid of this vector - Google Patents

Description

in DK 173763 B1 5 For maximal expression of foreign genes in microbiological systems, it is usually appropriate to use homologous regulatory elements within the expression vector. The efficiency of the expression ice (product formation) is assumed to be a function of and proportional to the strength of the promoter used. Furthermore, regulation of gene expression by nutritional factors under the control of the experimenter will provide yet another suitable manipulative tool. The glycolytic enzyme genes for yeast, i.e. those which encode pyruvic acid kinase (PyK) possess the above-mentioned valuable properties, viz. high expression (and thus, by interference, are very effective promoters) and susceptibility to regulation by components of the growth medium.

The invention thus relates to the isolation, products, and the use of yeast expression plasmids of DNA fragments corresponding to the 5 'non-coding regions of the regulated genes PyK in yeast. These fragments, which contain DNA sequences with strong transcriptional promoter activity, are called "promoters". These are ideal components of DNA vectors for the technical production of large amounts of proteins encoded by foreign genes under their transcriptional control. The foreign gene is hepatitis B surface antigen or parts thereof.

30

Furthermore, the invention relates to yeast expression plasmids which comprise a suitable terminator for forming a "cassette" of promoter-foreign gene terminator. The presence of the terminator increases the expression of the foreign DNA.

DK 173763 B1 2

An early attempt to express foreign DNA in yeast has failed (Beggs, J.D. et al., Nature (1980) 283: 285). IN

this report, the haemoglobin DNA (introduced together with its own promoter) was transcribed, but there was "similar" spotted 5 RNA. A number of explanations for this result are possible, e.g. an incorrect location for initiating transcription and / or poor ability causes the yeast cells to perform splicing of affected sequences (introns).

The PyK gene has been cloned but only by genetic complementation (no structural studies have been performed) (Kawasaki, G. and Fraenel, D.G., Biochem. Biophys. Res. Comm. (1982) 108: 1107). Other yeast promoters, e.g. for alcohol dehydrogenase I (Valenzuela, P. et al., Nature (1982) 298: 347 and Hitzeman, RA et al., Nature (1981) 293: 717) and phosphoglycerate kinase (Tuite, MF et al., EMBO J. (1982) 1: 603 and Hitzeman, RA et al., Science (1983) 219: 620) have been linked to foreign genes for the production of yeast expression, but no terminators have been used. . The present invention provides novel promoters for yeast expression systems, thereby providing the benefits of highly express promoters, combined with improved expression found with appropriately ligated terminators.

25

Published European Patent Application (EP 072 318) discloses the construction of a yeast expression system which, upon induction, expresses hepatitis B virus surface antigen (HBsAG) S protein under the control of the yeast 30 alcohol dehydrogenase I (ADH-I) promoter.

Thus, the present invention relates to a vector for expression in yeast and of the kind set forth in the preamble of claim 1.

DK 173763 B1 3

The vector is peculiar to that of the characterizing part of claim 1. The vector encodes hepatitis B virus (HBV) surface antigen (HBsAg) under the transcriptional control of a yeast PyK promoter. Terminators may also be appropriately attached. The expression vector typically has a yeast replication origin and a bacterial replication origin and is capable of replication in both cell types. The expression vector, used to transform yeast cells, will yield substantial amounts of the protein encoded by the foreign DNA segment. Expression of HBsAg under the control of yeast PyK promoter gives surprisingly high yield, cf. Example 4 and Table 1.

15

The invention will be explained by means of the drawing, in which fig. 1 nucleotide sequence of pyruvic acid kinase (PyK) gene, and 20 fig. 2 shows the structure of yeast expression plasmid containing PyK promoter region.

In principle, yeast expression plasmids have particular advantages, including those set forth below. Yeast can be grown on a large scale under well-known production conditions. By contrast, bacteria, grown as a large-scale culture, are exposed to the frequent "phage-out" problem.

Yeast also appears to have the same ability as animal or human cells to supply carbohydrate groups to newly synthesized proteins, an ability of bacteria not to possess. Since cDNA sequences are readily available, the problem of expression of genes having introns can be easily avoided.

DK 173763 B1 4

Furthermore, the invention relates to a method for expressing the yeast DNA coding segment contained in the vector, which method is characterized by the characterizing part of claim 6.

The vector in question comprises promoters with exceptionally high efficiency. A promoter is herein defined as a DNA segment which can function by initiating transcription of a connected DNA segment. Transcription is the synthesis of RNA (herein defined as messenger RNA or mRNA) that is complementary to one strand of the DNA attached to the promoter region. In eukaryotes, messenger RNA synthesis is catalyzed by an enzyme termed RNA polymerase II. The minimal essential elements of the promoter function are as follows: to provide a starting point for the initiation of transcription and to form a binding site to enable the current strand of DNA to be selected as a mold for messenger RNA synthesis.

Furthermore, a eukaryotic promoter functions to regulate the relative efficiency of transcription of coding segments under its control. An active promoter is one that produces synthesis of relatively large amounts of mRNA complementary to a strand of the associated DNA coding segment.

The structural correlations for promoter function have not yet been clearly established. A promoter segment can usually be identified as a region close to 30 at the 5 'end of a given structural gene. (The references to the 5 'and 3' ends of a gene will be understood to inject the corresponding respective ends of mRNA transcribed therefrom and these correlate with the NHc and -COOH end groups, respectively, of the encoded protein).

DK 173763 B1 5

Comparisons of the nucleotide sequences of promoters for different genes from different species have revealed only a few short regions of highly similar nucleotide sequence. Most notable for these 5 is the "TATA Box", which is a 5-10 nucleotide segment, generally located approx. 70-230 nucleotides in the direction of the site from transcription initiation, and with a sequence generally similar to TATAA. An overview of structural comparison appears in Breathnach, R. and Chambon, P., Ann. Rev.

Or Biochem. (1981) 50: 349. The TATA Box is believed to work when initiating transcription.

The foreign gene will be free of or substantially free of codons from the normal structural gene attached to the promoter. Usually, the foreign gene will be attached to a non-coding 3 'end of the regulatory region comprising the promoter, and thus be free of the amino acids at the N-terminus of endogenous gene naturally associated with the regulatory region.

Ie. that fewer than 3 codons (9 nucleotides) will be retained with the regulatory region when attached to the foreign gene.

The presence of the terminator sequence at the 3 'end of the 25 coding segment enhances the expression ice. The effect is generally similar to the addition of the rho factor to prokaryotic transcription systems, in which the rate of RNA polymerase release is increased to produce an increase in the rate of reinitialization of transcription. It will be appreciated that while the terminator sequences are not required for detectable expression of foreign DNA segments, it is preferable to associate them with elevated expression. The terminator region may be naturally associated with the same or different structural genes as the promoter region.

The most suitable DNA vector for PyK construction according to the invention is a shuttle vector. These vectors may alternate as a shuttle between a bacterial strain, e.g. E. coli, and yeast, because they have a bacterial replication origin and a yeast replication origin, see e.g. Ammerer, G. et al., Recombinant DNA, Proc. Third Cleveland 10 Symposium Macromolecules (Walton, A.G., ed.), P. 195,

Elsevier, Amsterdam (1981). A typical bacterial replication origin is e.g. derived from pBR322. The most suitable yeast replication origin is found in the extra-chromosomal genetic element known as the 2 mi-15 cron circle. In laboratory strains, 2 microplasmid DNA have been found to be present in approximately 50 copies per cell and maintained stable. An overview of this can be seen, for example. by Curr. Topics Micro. Imm. (1982) 96: 119.

This yeast plasmid has also been determined (Hartley, J.L. et al., Nature (1980) 286: 860).

Representative samples of the plasmids and host cells used in the structure of the invention are deposited with the American Type Culture Collection, 12301 Park-25 lawn Drive, Rockville, Maryland. Plasmid pPyK 9.1.1 and yeast cell transformants 2150-2-3 / pHBS-56 and 2150-2-3 / pHBS56PyK were deposited on February 18, 1983 and have been assigned ATCC registration numbers 40061 and 20666.

Many of the techniques, reactions and separation procedures mentioned in the following examples are well known in the art. All enzymes, unless otherwise stated, can be obtained from one or more commercial sources, such as New England Biolabs, Beverly, Massachusetts; Colla- DK 173763 B1 7 Borative Research, Waltham, Massachusetts; Miles Laboratories, Elkhart, Indiana; Boehringer Biochemicals, Inc., Indianapolis, Indiana and Bethesda Research Laboratories, Rockville, Maryland. Buffers and reaction conditions for 5 enzyme processes were selected on the recommendation of the supplier of each enzyme, unless otherwise indicated. Standard methods for other enzyme reactions, gel electrophoresis separations and E. coli transformation are described in Methods in Enzymology, (1979) 6j3. Transformation of 10 yeast protoplasts can be performed as described by Beggs, Nature (1978) 275: 104.

The strain of E. coli suitable for transformation includes X1776; K12 strains 294 (ATCC no. 31446); RR.1 and 15 HB101. Yeast strains XV610-8c with genotype (a ade2 ade6 leu2 lysi trpl canl) and GM-3C-2, genotype: (Leu2 Trpl

His4 CYC1-1CYP3-1) (Faye, G. et al., Proc. Natl. Acad.

Sci. (1981) 78: 2258) can typically be used for yeast transformations. However, it will be appreciated that virtually any 20 yeast species is suitable for transformation. Bacteria can be grown and selected according to a procedure described by Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1972). Yeast can be grown on the following media: 25 YEPD with 1% (w / v) yeast extract, 2% (w / v) peptone and (w / v) glucose; and, in the case of plate medium, 3% (w / v) agar.

YNB plus CAA contains 6.7 g of yeast nitrogen base (Difco Laboratories, Minneapolis, Minnesota), 10 mg of adenine, 10 mg of uracil, 5 g of casamic acids (CCA) (Difco), 20 g of glucose; and 30 in the case of plate media, 30 g agar per ml. liter. Selection of tryptophan prototrophy may be performed on plates containing 6.7 g of yeast nitrogen base (deficiency amino acid), supplementary with all growth conditions of the strain to be transformed, with the exception of tryptophan.

Example 17

Cloning of yeast pyruvic acid kinase gene 5

The pyruvic acid kinase gene was cloned by complementation. A yeast pyruvic acid kinase minus mutant was transformed with a portion of recombinant YEp24 plasmids containing yeast genomic DNA. Yeast strains S288C {genotype: 10 SUC2, mal, ga! 2, CUP1) and PyK 1-5 (genotype a, noble, leul, met! 4, ura3, pykl-5) were provided from the Yeast Genetic Stock Center, Department of Biophysics , University of California, Berkeley. The yeast genome bank used consists of a partial Sau3A preparation of total DNA from the strain S288C cloned into the BamHI site of "shuttle" vector YEp24. The vector YEp24 contains pBR322 sequences for selection and growth of bacteria, the yeast URA3 gene for selection in yeast, and an EcoRI fragment of yeast 2 μ circle to assure plasmid replication and segregation in yeast. The portion comprises sufficiently independent recombinant plasmids to represent the entire yeast genome.

The pykl-5 strain is unable to grow on medium containing glucose or lack of uracil due to mutations in this strain at the PyK1 and URA3 sites, respectively. Transformation of this strain with YEp24 genomic library and selection for transformants capable of growing on a medium lacking uracil and containing glucose selects such cells that have acquired YEp24 containing the pyruvic acid kinase gene. Transformation of 3.5 x 10 9 pykl-5 yeast cells with 10 µg of YEp24 recombinant plasmid DNA yielded 5 independent transformants which grew in the absence of uracil and in the presence of glucose.

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Characterization of the inserted DNA in these transformants by restriction enzyme analysis indicated that they contained overlapping DNA inserts. The focus is on a single transformant, pPyK 9.1, which contained a 7.0 kb insert. The pyruvic acid kinase gene was localized within this insert by determining which insert-specific restriction fragments hybridized to an mRNA of ca. 1.7 kb, which is expected for pyruvic acid kinase mRNA. Localization of the PyK gene was confirmed by subcloning appropriate regions of the inserted DNA and observing complementation of function in pykl-5 mutant. A subclone pPyK 9.1.1 containing PyK gene on a 4.4 kb insert was sequenced and used to express plasmid structures.

EXAMPLE 2

Sequence of yeast pyruvic acid kinase gene 20

A total of 2885 nucleotides of the PyK gene have been sequenced, comprising 1497 single open chain nucleotides, 911 nucleotides of the 5 'untranslated region and 477 nucleotides of the 3' untranslated region (see Fig. 4).

The gene encodes a polypeptide of 499 amino acids to give a monomeric molecular weight of 54,608 daltons, which is in good agreement with the expected value for yeast PyK. The amino acid composition derived from the nucleotide sequence also corresponds closely to that measured for the isolated yeast protein. The nucleotide sequence predicts a carboxy-terminal valine found for yeast pyruvic acid kinase.

Example 17

Yeast Expression Plasmid Structure Using Pyruvic Acid Kinase Promoter Region 5

Two different structures were prepared: pHBS16 PyK and pHBS56 PyK. The procedure is that of FIG. 5 illustrated.

Plasmid pPyK 9.1.1, which contains the yeast PyK gene cloned 10 in pBR322, was reacted with XbaI and the protruding ends were filled with deoxynucleotides by DNA polymerase I. The product was reacted with BamHI to finally isolate a 912 bp BamHI blunt fragment containing PyK promoter and 8 bases from PyK coding region. This fragment 15 was alloyed to plasmid pHBS-6 (contains the HBsAg gene / in which the 5 'non-coding region has been removed, cloned into pBR322), pre-digested with NcoI, filled in by DNA polymerase and reacted with BamHI. After transformation of E. coli, pHBS-6PyK was isolated. This plasmid contains PyK promoter with codons for 3 extra amino acids, fused in phase with HBsAg coding sequence, ATGTCTAG CATG.

I_I 1_I

25 pHBS-6PyK was reacted with BamHI for completion and partially reacted with EcoRI to isolate the 1750 bp BamHI-EcoRI fragment containing PyK promoter fused to the HBsAg gene. This 1750 bp fragment was ligated to the large 30 fragment obtained after reaction of pHBS-16 (ATCC register No. 40043, plasmid, described in U.S. Patent Application No. 442,687, entitled "Adenovirus Promotor in Yeast", with Bam HI and Eco RI and used to transform E. coli, the yeast expression plasmid pHBS-DK 173763 B1 11 was obtained 16 PyK. This pHBS-16PyK was digested with Sphl and Xba bp of pBR322, the PyK promoter, and 100 bp of the 5f region of the HBsAg gene) were isolated This 1200 bp Sphl-5 XbaI fragment was ligated to a 1070 bp XbaI-Sphl fragment (isolated from pHBS-56 ) containing the 3 'end of the HBsAg gene and the ADH-1 terminator. After reaction with Sphl, a SphI-Sphl 2300 bp fragment (cassette) containing PyK promoter, HBsAg gene and ADH-1 terminator was isolated.

This cassette fragment was cloned into pHBS-56, which was previously reacted with SphI. Yeast expression plasmid pHBS-56 PyK was obtained. This plasmid was used to transform the yeast strain AB102 (see Example 2) or the strain 2150-3 (see Example 2).

EXAMPLE 4

Synthesis of HBsAg in Yeast under PyK Promoter Control 20 100 ml Cultures of strain AB102 containing plasmid pHBS-56 PyK were grown to optical density at 650 nm of 1-2. Cell-free lysate was prepared by stirring with glass beads and removing cell debris by centrifugation. HBsAg was measured by Abbott Ausriall radioimmunoassay and the protein concentration was determined by the Coomasse blue binding method. The results are shown in Table 2. They show that PyK promoter is at least twice more effective than ADH-1 promoter for expressing the protein product in yeast.

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Similar results were obtained by using the yeast strain 2150-2-3 instead of the strain AB102 and repeating Example 4.

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I- I

5 IN TABLE 1: Synthesis of HBsAG in yeast | I-1

j (a) from pHBS-56 (control, ADH-I promoter) J

In Spec. IN

IExp # sAq protein activity | 10 I (pg / ml) (mg / ml) (pgsAg / mg protein) |

I I

I 1 8, 2 24 0, 34 | I 2 7, 2 24 0, 32 |

1 3 4, 7 27 0.23 I

15 I I

In (b) from pHBS-56 PyK (PyK promoter) | In Spec. [

[Exp # sAg Protein Activity I

I (pg / ml) (mg / ml) (pgsAg / mg protein) |

20 I I

j 1 18 2.5 0, 68 I

] 2 10, 6 22 0, 48 I

j 3 15, 2 27 0, 56 | i__________i 25

Similar results were obtained by using the yeast strain 2150-2-3 instead of strain AB102 and repeating Example 4.

Claims (9)

A yeast expression vector consisting of a foreign DNA segment under the control of a yeast pyruvic acid kinase promoter, characterized in that the segment is oriented correctly for transcription and has fewer than three codons of yeast pyruvic acid kinase at the 5 'end of 10 said foreign DNA encoding hepatitis B surface antigen or a portion thereof. Vector according to claim 1, characterized in that it comprises a terminator attached to the 3 * end of the foreign DNA segment. Vector according to claim 1, characterized in that it comprises yeast 2 micron plasmid DNA or part thereof. 4. Plasmid pHBS-56 PyK having ATCC number 20666. A method for expressing a DNA coding segment in yeast, characterized in that (a) the coding segment is inserted into a yeast expression vector comprising a DNA segment containing yeast pyruvic acid kinase promoter which has fewer than three codons from the 5 'end of the yeast pyruvic acid kinase, which promoter is attached to the 5' end of the inserted 30 DNA coding segment and oriented so that the transcription initiated in said promoter comprises the coding segment, so that a coding segment expression vector arises in which the DNA segment DK 173763 B1 encodes hepatitis B surface antigen or a portion thereof. (b) transforming the yeast cells with the coding segment expression vector. Method according to claim 5, characterized in that the yeast expression vector further comprises a terminator attached to the 3 'end of the insert. DNA coding segments. The method of claim 5, characterized in that the yeast expression vector further comprises a bacterial cell replication and is capable of replicating a bacterial cell. Process according to claim 6, characterized in that said terminator comprises alcohol dehydrogenase I terminator from yeast. 20 Method according to claim 6, characterized in that said terminator consists of PyK terminator from yeast.