NZ214422A

NZ214422A - Dna vector, process for making proteins and transformed cells

Info

Publication number: NZ214422A
Application number: NZ214422A
Authority: NZ
Inventors: A Sledziewski; E Chlebowicz-Sledziewska
Original assignee: Boehringer Ingelheim Int
Priority date: 1984-12-04
Filing date: 1985-12-03
Publication date: 1988-02-12
Also published as: JPS61181385A; EP0184163A2; ES549526A0; DK559585D0; HUT39775A; GR852891B; FI854781A0; ES556975A0; NO854864L; EP0184163A3; DK559585A; ES556976A0; ZA859240B; HU196454B; DE3444495A1; SU1364241A3; ES8802075A1; PT81594A; ES8801705A1; FI854781A

Description

<div class="application article clearfix" id="description"> 2 1442 Priori;-/ - r ."€^' ^^3^, .... ...i,ed:^r^:g$j V • oo: • ^ i w j.'Zay.. c.alu2 i/aj. Pub/i cation D->fe: .r?.FEB1988 P.O. Jouma;. ' PATENTS FORM NO. 5 NEW ZEALAND PATENTS ACT 195 3 COMPLETE SPECIFICATION "MULTI-COPY YEAST VECTORS" £, WE BOEHRINGER INGELHEIM INTERNATIONAL GMBH a German Body Corporate of D-6507 Ingelheim am Rhein, Federal Republic of Germany, hereby declare the invention, for which-I-/we pray that a patent may be granted to -ste/us, and the method by which it is to be performed, to be particularly described in and by the following statement -1- 2 14422 - 2 - The present invention relates to recombinant multicopy yeast vectors, to their use, to a process for preparing these vectors and to a method of multiplying these vectors. 5 The preparation of proteins in suitable host organisms by genetic engineering conventionally involves inserting heterologous DNA which codes for the desired proteins into the double-stranded DNA of 10 a suitable cloning vector which is then introduced into a suitable host organism. The replication system of the host organism then reproduces the inserted DNA fragments along with the DNA sections of the original host. Not only is the DNA replicated 15 but also, provided the plasmid contains a suitable reading frame and promoters, the protein encoded by it is expressed. The protein yield naturally depends on the efficiency of the replication system of the host organism but also on the available 20 quantity of expressible DNA material. Many plasmids exist as only a single copy per cell; a few exist in more than one copy per cell. The plasmid ColEl exists, for example, in 10 to 20 25 copies per E. coli chromosome. Plasmids derived from ColEl can be multiplied by adding protein synthesis inhibitors but, obviously, the protein cannot be accumulated in this way. Alternatively the copy number of so-called "runaway plasmids" 30 may be multiplied by a temperature increase. However, the protein yield does not depend only on the number of copies of the plasmids but also on the stability of the plasmids themselves. In 35 the case of vectors suitable for yeast, plasmids are known which can be multiplied to high copy numbers but which are unstable; others are extremely 2 144 - 3 - stable bat exist only in small numbers of copies per cell. One aim of this invention was to prepare a vector 5 system suitable for yeast which could be kept stable and used with a satisfactory number of copies. Q Many plasmids with heterologous DNA can only be used for the preparation of proteins by genetic 10 engineering under selecting conditions since otherwise the non-transformed organisms would become too numerous. These selecting conditions can frequently only be established by using antibiotics. These substances make preparation more expensive and ; 15 the disposal of the fermentation liquors containing -2 antibiotics presents problems (danger of developing -"j antibiotic resistance) . y- a ; A further object of this invention was therefore 20 to prepare a recombinant multi-copy yeast vector which could be cultivated in a stable manner under non-selective conditions. In one aspect, our invention provides a recombinant 25 stable multi-copy yeast vector, wherein said vector contains a stabilising function and a regulatable promoter by means of which the stabilising function can be controlled. Preferably the stabilising function is a yeast centromere. 30 The literature references cited in the accompanying bibliography illustrate the prior art and describe experimental procedures and products applicable to the present invention. Where necessary, they 35 are cited by a cross-reference by number. ■ • 2 1442 There are various types of vectors which can be used for the expression of heterologous genes in yeast (1). One class of yeast vectors consists of so-called "yeast integrating plasmids" (Yip) which are normally made up of bacterial plasmids such as pBR322, fragments of yeast chromosomal DNA and a selection marker. These vectors transform yeast in very low yields (1 to 10 transformed cells per 1 meg of plasmid DNA). The transformed phenotype is obtained by integration of the Yip plasmid into the yeast chromosome (2,3). It is known that some fragments of yeast or other eukaryotic DNA Yip plasmids impart the property 15 of replicating themselves extrachromosomally (4-8). Vectors which contain the ARS element (autonomously replicating sequence) are referred to as YRp (yeast replicating plasmids) or simply ARS plasmids. Investigations of the mitotic stability of the 20 YRp plasmids have shown that, even under selective growth conditions, only a few cells (5-25%) contain the plasmid (4, 5, 7, 9). However, the number of copies of these plasmids varied from about 20-50 copies per cell (7,10). 25 It is also known that the presence of so-called centromere sequences on a yeast vector frequently improve its stability. However, the centromere function does not improve the number of plasmid 30 copies per cell. The mitotic stability of the YRp plasmids can be improved by adding fragments of the yeast DNA, assumed to constitute the functional elements of 35 a centromere (CEN) (11, 12). These plasmids, known as YCp (yeast centromere plasmids) are present in about 80% of the cells grown under selective i. 1 4 4 z 2 - 5 - conditions in 1 to 2 copies per cell (11, 12). The yeast Saccharomyces cerevisiae contains an endogenous DNA plasmid which is known as the "2-micron 5 circle"; it is mitotically stable and present in 20 to 50 copies per cell (13). The 2-micron circle carries its own amplification system which enables the plasmid to replicate more than once per cell cycle (14, 15). Many different yeast vectors have 10 been constructed on the basis of the 2-micron circles; the majority are mitotically stable up to 60-80% on non-selective medium with a copy number of 20-50 per cell (14 to 16). Thus, they have partly lost the stability of the 2-micron circle. 15 The present invention is based on the discovery, inter alia, that the yeast centromere function (CEN) can be regulated by transcription originating from a regulated yeast promoter. 20 It has been demonstrated that the yeast centromere DNA no longer mitotically stabilises YRp plasmids if it is transcribed by a strong promoter (17). In the present invention, a CEN sequence, preferably 25 a CEN3 sequence, was placed in front of a regulatable yeast promoter, preferably alcohol dehydrogenase II promoter; the YCRp plasmid family (yeast centromere regulated) was obtained. 30 The expression of the yeast alcohol dehydrogenase-II gene (ADH2) is repressed in the presence of glucose and reactivated in the presence of non-fermentable carbon sources such as ethanol (18). It has been demonstrated that the 5'-flanking sequences of 35 the ADH2 gene, when correctly ligated to another gene, can confer glucose repression on such a gene (19). By connecting the CEN3 sequence in front 2 1442 2 ' - 6 - of the 5' flanking regulation sequences containing the ADH2 promoter (referred to as the ADH2 promoter for abbreviation) it will be possible to control the expression of the ADH2-CEN3 fusion by simply 5 changing the carbon source in the medium. If the yeast transformed with an ADH2-CEN3 fusion plasmid (YCRp) is cultivated in the presence of glucose, the ADH2 promoter is repressed (ADH2-0FF), and CEN3 stabilises the YCRp plasmid mitotically (CEN3-0N). 10 When the carbon source is changed to ethanol the ADH2 promoter is derepressed (ADH2-ON) and expression through the CEN3 region is blocked (CEN3-OFF). As a result, the YCRp plasmids are no longer stable. At this stage, it will be possible to amplify 15 the YCRp plasmids in yeast cells up to the desired copy number. After amplification, these YCRp plasmids may be stabilised by simply changing the medium from ethanol to glucose by means of the now functional CEN3 sequence. 20 The invention is not restricted only to the CEN3 sequence as a stablising element and to the ADH2 gene as a regulatable element; it is also possible to have other stabilising elements such as, for 25 example, CEN6 and CEN11, if they are properly fused with tightly regulatable yeast promoters. Depending on the promoter, regulation can then be achieved by a number of possible factors such as, for example, choice of carbon source, heat shock, oxygen, heme, 30 or phosphates. It will be clear that our invention may be used to multiply a heterologous DNA coding for a desired heterologous protein. In such a process: 35 a) a heterologous DNA coding for a desired protein is inserted in suitable manner into a vector o 2 1442 - 7 - according to the invention; b) the vector is transformed into a suitable yeast host organism which contains wild-type copies 5 of the 2-micron circle plasmid, c) the host is initially cultivated on a medium which represses the regulatable promoter, 10 d) then the host is cultivated on a medium which derepresses the regulatable promoter, e) the vectors are isolated from the cells and purified and 15 f) the heterologous DNA is isolated from the vectors and purified. Preferably between steps (d) and (e) the host is again transferred to a medium which represses the regulatable 20 promoter and is cultivated. Likewise we may multiply the vector according to the invention by a process wherein 25 a) the vector is transformed into a suitable yeast host organism which conta of the 2-micron circle plasmid, yeast host organism which contains wild-type copies b) the host is initially cultivated on a 30 medium which represses the regulatable promoter, c) then the host is cultivated on a medium which derepresses the regulatable promoter and 35 d) subsequently the host is again transferred to the medium which represses the regulatable promoter and is cultivated. 2 1 44Z2 - 8 - The accompanying drawings are intended to illustrate but not to limit the invention. Figure 1 schematically shows the restriction 5 and genetic map of the pBC3Tl and ADH2 BS plasmids. It also shows the preparation of the YCRp2 plasmid. Figure 2 schematically shows the restriction and genetic map of the JDB207 plasmid. It also shows the preparation of the YCRp3 and YCRp4 plasmids. 10 Figure 3 shows an assay for determining the copy number of YCRp plasmids. All the DNA restriction and metabolism enzymes used were obtained from New England Biolabs and 15 from Bethesda Research Laboratories. The enzymes were used under the conditions and in the buffers recommended by the retailers. ATP and the deoxy-nucleotide triphosphates were obtained from SIGMA; the DNA linkers were obtained from New England 20 Biolabs. The purification of covalently closed circular plasmid DNA from E. coli and the transformation of E. coli were carried out using methods which 25 have already been described (20, 21). "E^coli-miniscreens" were used as described (22). The transformation of the yeast was basically carried out according to known methods (2) but with the 30 following modifications: 200 ml of cells with a content of 2 x 107 cells/ml were purified by washing with 25 ml of H20 and centrifuging. These cells were treated with 10 35 ml of 1 M sorbitol, 25 mM EDTA (pH 8) and 50 mM dithiothreitol solution for 10 minutes at 30°C and then washed with 10 ml of 1 M sorbitol. The I Q 10 2 1 442 - 9 - pelleted cells were then carefully re-suspended in 10 ml of SCE (1M sorbitol, 0.1 M sodium citrate pH 5.8 and 0.01 M EDTA) and treated at 30°C with 1 mg of zymolyase 5000 (Kirin Brewery). 80% sphero-plasting was carried out by adding 0.1 ml of the suspension to 0.9 ml of a 10% SDS solution; measurement was effected at Absggg using a cell solution before the addition of the enzyme as a 0 percent comparison (lysis in 10% SDS resulted in a drop in the AbSg0Q). The cells were then washed three times with 10 ml of 1 M sorbitol, once with 1 M sorbitol, 20 mM CaCl2 and 10 mM Tris-HCl (pH7.4) and then re-suspended in 1 ml of the same solution. 5 to 15 15 meg of the purified plasmid DNA were then added and carefully mixed for 15 minutes with 0.1 ml of the re-suspended cells. 1 ml of a solution containing 20% (w/V) of polyethyleneglycol 4000 (Merck), 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5) 20 was added within 15 minutes with careful mixing. The cells were then removed by centrifuging and incubated in 0.2 ml of SOS (1 M sorbitol, 33.5% (v/v) YEPD Broth and 6.5 mM CaCl2) for 20 minutes on 30°C. 0.1 ml of this suspension was then spread 25 to a petri dish which contained 20 ml of "bottom-agar" (182 g of sorbitol, 20 g of glucose, 0.7 g of YNB and 30 g of Difco agar per 1 litre of H20) and treated with 10 ml of "top-agar" at 50°C (same composition as the bottom-agar but additionally 30 containing 1 ml of adenine (1.2 mg/ml), 1 ml of uracil (2.4 mg/ml) and 1 ml of a "-trp drop-out mix" per 50 ml of "bottom-agar" ("-trp drop-out mix" contains the following amino acids per 100 ml of H20: 0.2 g arg, 0.1 g his, 0.6 g ile, 0.6 g 35 leu, 0.4 g lys, 0.1 g met, 0.6 g phe and 0.5 g + 3 thr). This Trp selection yielded 10 yeast transformants per meg of plasmid DNA. A 2 1442 - 10 - The stability of the plasmids in yeast was tested by diluting cells in water after selective or nonselective growth and spreading them on YPD plates (non-selective)- After 30 hours growth at 28°C 5 these plates were replica plated on YNB+CAA plates (TRP+ or LECJ+ selection). The percentage plasmid stability was determined by dividing the number of colonies which had grown selectively by the number of colonies which had grown non-selectively, 10 multiplied by 100. The copy number of the plasmids was determined by Southern analysis (23) . The entire DNA of the SHU 32 (YCRp) transformants which had grown in 15 selective medium was digested with EcoRI restriction endonuclease and fractionated by agarose gel electrophoresis. After being transferred to nitrocellulose filters the DNA was hybridised with YIp5 plasmid DNA radioactively labelled by nick translation 20 (YIp5 is pBR322, which contains the yeast URA3 gene (3)). After exposure on X-ray film at least 2 bands were observed: one showed the single 0RA3 chromosomal copy, whilst the other(s) is (are) the YCRp plasmid fragment(s). A DNA probe of SHU 25 3 2 transformed with the YCP plasmid served as a further control; this made it possible to compare the copy numbers of YCP and YCRp centromere plasmids directly. In some tests, serial dilutions of the complete yeast DNA were developed on the gel so 30 that it was possible to make a direct comparison between the intensity of the YCRp bands and that of the control plasmid bands which are known to occur in only 1 to 2 copies per cell. 35 For transforming bacteria, the E.coli strain RR1 was used (F . pro , leu , thi-, lacY, rpsH, hsdR, hsdM)(21). The yeast Saccharomyces cerevisiae 2 1442 - n - strain SHU 32 (leu2, trpl, ura3; kindly supplied by Dr. A. Hartig, University of Vienna) was used for the yeast transformations. Obviously, other yeast strains may also be used. 5 The LB medium used was described by Miller (27) , but with the addition of 30 mcg/ml of ampicillin (SERVA) after the medium had been autoclaved and cooled. The yeasts were cultivated on the following 10 media: YP (1% yeast extract, 2% peptone and a carbon source, either 5% glucose or 3% ethanol), YNB+CAA (0.67% yeast-nitrogen base w/o amino acids) (Difco), 0.5% Difco Casamino acids (CAA) and a carbon source, either 5% glucose or 3% ethanol). 15 For TRP+ selection YNB+CAA was supplemented with -trp drop-out solution (1 ml of the 50 x solution per each 50 ml of medium), for LEU+ selection -leu drop-out solution was employed in the same way. The yeast media were solidified by addition of 20 2.5% of Difco agar. In Figure 1 two plasmids, pBC3Tl and ADH2Bs,are shown. Information about the construction of both these plasmids has been published (19, 28) . 25 pBC3Tl contains a portion of pBR322 (19) with the D ampicillin resistance gene (Ap ) and the E.coli origin of replication (ORI) for selection and stable growth in E.coli. The plasmid also contains the TRPl gene on an EcoRI 9 EcoRI 1.45 kb fragment 30 which comes from chromosome III of yeast (30) . This gene allows for selection in trpl- yeast strains and therefore can be used to isolate yeast clones containing this plasmid. On the same DNA fragment the ARS1 (autonomously replicating sequence) element 35 is present. The ARS1 element allows the DNA to replicate autonomously in yeast and be maintained as a plasmid (30) . The pBC3Tl plasmid also contains 1442 2 - 12 - the CEN3 sequence on a Hindlll - BamHI 2.0 kb fragment which originates from yeast chromosome III (31). Plasmids containing CEN3 sequence are mitotically stable and present in 1 to 2 copies per cell. 5 The ADH2BS plasmid is pBR322 with a 2.0 kb BamHI -Sau3A fragment of yeast chromosomal DNA containing 'ADH2 gene cloned into the unique BamHI site (2B). Ligation between the BamHI and Sau3A "sticky" ends 10 regenerates the BamHI restriction site, so that the ADH2 gene can be cut out from the ADH2BS plasmid with a single BamHI digestion. It is known from the published DNA sequence of 15 the ADH2 gene and its 5' flanking regions that a convenient EcoRV restriction site is present at the +67 position (where +1 is the A in the ATG start codon). This EcoRV site, together with the BamHI site located at position -1207 was used to 20 isolate the ADH2 promoter with all its 5'-flanking regulatory sequences. Typically 5 meg of ADH2BS plasmid DNA was completely digested with EcoRV (Figure 1), purified by phenol 25 extraction and ethanol precipitation, blunt-end ligated with 2 meg of Bglll phosphorylated linker (5'-CAGATCTG-3) (Biolabs), restricted again with BamHI and Bglll and after preparative agarose gel electrophoresis, a 1300 bp fragment was isolated. 30 This fragment contained the intact ADH2 promoter with all regulatory sequences and a small piece of ADH2 coding region (coding for the first 22 amino acids). 35 5 meg of pBC3Tl plasmid DNA was completely digested with BamHI endonuclease, purified by phenol extraction and ethanol precipitation and dephosphorylated is • - / r ■ o o 2 1 4422 - 13 - with calf intestinal phosphatase (CIP) by known methods (33). After another phenol extraction and ethanol precipitation 100 ng of BamHI digested and dephosphorylated pBC3Tl plasmid DNA was ligated 5 with 200 ng of 1.3 kb BamHI-Bglll fragment of ADH2 promoter, for example with T4-DNA ligase. Competent E.coli RR1 cells were subsequently transformed with one fifth of this mixture. Ampicillin resistant colonies were screened by colony hybridization ^ 10 method using the ADH2 1.3 kb BaraHI-Bglll fragment, | radioactively labelled by nick-translation, as 1 a probe. Plasmid DNA was prepared from 12 colonies ■1 | which gave a positive signal and digested with r a variety of restriction enzymes to identify the E 1 15 clones with the inserted ADH2 promoter cloned in a suitable orientation so as to allow transcription from the ADH2 promoter through the CEN3 sequence. Such a clone was identified and the plasmid named YCRp2. The restriction map of the YCRp2 plasmid 20 is given in Figure 1. To determine the copy number and stability of the plasmid in yeast, the yeast strain SHU 32 (trpl , leu2~, ura3~) was transformed with YCRp2 plasmid 25 DNA. The TRP transformants were isolated and assayed for their stability and copy number. The data from these experiments are presented in TABLE 1. The YCRp2 plasmid stability was assayed after trans-30 formants were grown in media with different carbon sources. When YCRp2 transformants grown only on glucose medium, the stability of the plasmids resembled that of other YCp plasmids (about 80%) and the copy number was low (1 to 2 copies per cell) (compare 35 Examples 1 and 3 from TABLE 1, columns 1 and 2 from Figure 3). This was expected because when glucose is present the ADH2 promoter is repressed j 2 1 442 - 14 - (ADH2 - OFF) and CEN3 sequence can function normally (CEN3 - ON). However, when YCRp2 transformants were grown on 5 medium containing ethanol, the stability of the plasmid decreased dramatically (to about 36%) but the copy number increased to 5 to 10 per cell. YCp plasmids under the same conditions were still very stable and present in low copy number (compare 10 Examples 2 and 4 in TABLE 1). This shows how transcription of the derepressed ADH2 promoter (ADH2 - ON) blocks the CEN 3 sequence (CEN3 - OFF) and as a result YCRp2 plasmid behaves like a YRp plasmid (very unstable but with high copy number). 15 Surprisingly, the stability of the YCRp2 plasmid was even greater than before when the medium was changed back to glucose. The stability on this medium was about 95% and the copy number remained 20 constant at 5 to 10 copies per cell of Example 5 in TABLE 1, column 3 in Figure 3). Glucose again represses the ADH2 promoter (ADH2 - OFF) and thus derepresses the CEN3 function (CEN3 - ON). 25 During cultivation on ethanol, however, the yeast itself could not accumulate more copies of the YCRp2 plasmid. Even after 50 generations on YNB+CAA ethanol medium, the copy number never exceeded 8 to 12 copies per cell (cf. Example 6 in Table 30 1; column 4 in Figure 3). Table 1 below shows the stability and copy number of data of the YCRp2 plasmid in yeast strain SHU32. < Q O G 9 Table 1 Exp. Strain Plasmid Carbon source medium in the % Plasmid Stability Copy w b> number 1 SHU 32 PBC3T1 Glucose 80 % 1 - 2 2 SHU 32 pDC3Tl Ethanol 76 % 1 - 2 3 SHU 32 YCRp2 Glucose 78 % 1 - 2 4 SHU 32 YCRp2 Ethanol c) Glucose 36 % 5 - 10 5 SHU 32 YCRp2 Ethanol — 95 % 5 - 10 6 SHU 32 YCRp2 Ethanol —* Glucosed' 95 % 8 - 12 a) % plasmid stability was determined as described after 25 generations on non-selective medium. 1 b) Copy number was determined after 12 generations on selective medium. c) The strain had been cultivated for 12 generations on ethanol-selective medium, then transferred to the glucose-selective medium for 12 generations and an inoculum of this culture was taken in order to determine the stability and copy number. d) The strain was cultivated as in c) but for 50 generations on ethanol. I\g ' K) NJ 2 1442 - 16 - preparation of the YCRp3 and YCRp4 plasmids The preparation of the YCRp3 and 4 plasmids is diagrammatically shown in Figure 2. References 5 to the cloning vector pJDB207 have already been published (16). pJDB207 contains a bacterial pATl53 plasmid (34) with the ampicillin (Ap ) and tetracycline resistance genes (TetR) and the ColEl replication origin (ORI) for selection and stable growth in 10 E. coli. The plasmid also contains a 2.7 kb EcoRI/EcoRI fragment of 2-micron DNA. Both the original 2-micron replication site (13), which puts the vector in the position of replicating extrachromosomally in yeast and manifesting itself, and the REP3 locus 15 of 2-micron DNA are located on this fragment. This locus is an important element of the 2-micron amplification system and must be present in cis configuration in order to make the plasmid capable of amplification. Other necessary elements such 20 as REPl and REP2 are controlled by wild-type copies of the 2-micron DNA in trans orientation (14, 15). The selection marker LEU2, originating from the yeast chromosome III, was cloned into the Pstl restriction site of the above-mentioned 2-micron 25 DNA fragment after some modifications. This LEU-2-d gene enables selection on leu2~ yeast mutants and complements the leu2~ mutation only when it is in a "multi-copy vector" (16). In order to prepare the YCRp3 and 4 plasmids, 5 meg of YCRp2 DNA were digested with Bglll and BamHI restriction endonuclease. After preparative agarose gel electrophoresis, a 4.2 kb Bglll/BamHI fragment was isolated. This fragment contained the yeast TRPl gene and the ADH2-CEN3 fusion. 5 meg of the pJDB207 plasmid DNA were completely digested with 30 O 35 2 144 ■ - 17 - BamHI and purified by phenol extraction and ethanol precipitation and dephosphorylated with calf intestinal phosphatase as described (33). 100 ng of the pJDB207 plasmid DNA thus obtained were then ligated with 5 200 ng of the 4.2 kb Bglll/BamHI YCRp2 fragment. E. coli RR1 cells were transformed with one fifth D of this mixture and Ap colonies were isolated. -> All the colonies were checked by colony hybridisation using a nick-translated 4.2 kb fragment of YCRp2 10 as a probe. The plasmid DNA was isolated from the positive colonies shown and a restriction map of these plasmids was produced. Since the Bglll/BamHI fragment can be cloned in two different orientations into the BamHI cutting site of the pJDB207, two 15 plasmids were produced: YCRp3 and YCRp4 (Figure 2) . Copy number and stability of the YCRp3 plasmid 20 It was found that the orientation of the cloned ADH2-CEN3 fusion has no effect on the stability or copy number of the YCRp plasmid in yeast. All the results were obtained with the YCRp3 plasmid, but the YCRp4 plasmid behaved in a similar manner. 25 It should be noted that the YCRp3 plasmid contains two yeast marker genes: LEU2-d and TRPl. The TRPl gene complements a trpl" mutation in yeast even when present in only one copy per cell, whereas 30 the LEU-d gene complements a Leu2~ mutation only when it is on a "multi-copy plasmid" (50-100 copies per cell). This system makes it possible to check the copy number by simple spreading onto selective medium. All LEU+ cells must contain at least 50 35 plasmid copies per cell, whilst TRP+ cells contain at least one copy of the plasmid. It should also be mentioned that the SHU 32 strain is cir+, i.e. 2 1442 - 18 - — it carries wild-type 2-micron DNA, which supplies the YCRp3 vector with two further components of the 2-micron amplification system: REPl and REP2. The fully active 2-micron amplification system 5 requires 4 loci: REPl and REP2 in trans and REP3 and ORI in cis. The yeast strain SHU 32 (cir+, trpl", leu2~, ura3~) was transformed with YCRp3 plasmid DNA, TRP+ transit) formants were isolated and tested on leu- and ura". All the transformants which were tested were leu" and ura- and showed that the YCRp3 plasmid is present in a low copy number. This result was not surprising since only glucose was used as a carbon source; 15 CEN3 should therefore be fully operational. It is noticeable that the CEN3 function overcomes the 2-micron amplification system and stabilises the copy number of the plasmids at 1-2 per cell (Example 2 in Table 2; column 8 in Figure 3). 20 The stability of YCRp3 in the yeast strain SHU 32 when grown on glucose is also characteristic of a YCp plasmid (about 80%). However, when the SHU 32 transformed with YCRp3 25 was cultivated on selective -leu ethanol medium, the stability of the plasmids as measured by the number of leu+ colonies decreased to about 65%. It is assumed that the CEN3 sequence is transcribed in the presence of ethanol by the derepressed ADH2 30 promoter, with the result that the CEN3 function is blocked. With the selection for LEU+ and nonfunctional centromeres, the 2-micron amplification system takes over the control and amplifies YCRp3 to a high copy number. SHU 32 transformants become 35 LEU and the stability of the YCRp3 plasmids under these conditions is characteristic of a 2-micron chimeric plasmid (cf. Examples 1 and 4 Table 2). Q 2 1442 2 - 19 - - This expectation was confirmed after the SHU 32 transformants, having been grown on ethanol, were cultivated on glucose and the copy number of the plasmid was determined. The data appearing in 5 columns 9-11 in Figure 3 clearly show that the copy number of the YCRp3 plasmid rose to approximately 100 per cell. In addition, the plasmid was very stable (more than 90%!) as was demonstrated by the number of leu+ colonies. Table 2 below shows the stability and copy number data of the YCRp3 plasmid in the yeast strain SHU 32. 10 i_a. -——=-~v vmmBomw 0 0 (o Table 2 Exp. Strain Plasmid Carbon source in the medium % Plasmid stability Copy « b) number LEU+ TRP+ 1 SHU 32 pJDB207 Glucose 72 100 2 SHU 32 YCRp 3 Glucose not growing 85 % 1 - 2 d.c) 3 SHU 32 YCRp3 Ethanol 65 % 85 % n. 4 SHU 32 YCRp3 Ethanol-KSlucose^ ^ 98 % 99 % 100 a) ft plasmid stability was determined as described after 25 generations on non-selective medium. b) Copy number was determined after 12 generations on selective medium. c) Not determined. d) The strain was cultivated as in c) but for 50 generations on ethanol. ' K N) 2 1 44 - 21 - The preparation of plasmids with centromere-regulated functions, as has been demonstrated for the YCRp plasmids, is of great value both for the multiplication of heterologous DNA and also for the expression 5 of heterologous proteins in yeast. If, for example, heterologous DNA is inserted into a multi-copy yeast vector in a suitable manner, according to the invention, it can be multiplied 10 with the vector up to the required concentration with the aid of the process according to the invention (alteration of the factors which influence the regulatable promoter, e.g. in the case of the ADH2 promoter, changing the carbon source). 15 The vectors according to the invention are of even greater value when heterologous DNA with a suitable expression system for the expression of heterologous proteins in the correct orientation is built into 20 the vectors. The efficiency of a fermentation process is critically dependent on the nature of the expressed protein. If, for example, a protein is expressed which is 25 toxic to the host organism above a certain concentration the entire fermentation process may become ineffective if the quantity of protein cannot be controlled directly or indirectly. 30 The vectors according to the invention make it possible to adjust the copy number of the expression plasmid simply by changing the factors which influence the regulatable promoter, e.g. changing the carbon source, to a low, medium or high value and to stabilise 35 the copy number at the level selected. With these vectors it is even possible to obtain a product which is toxic to the host system by keeping the * © 2 1442 - 22 - copy number of the expression vectors in a fermentation mixture low for several generations and keeping the individual plasmids stable. Once sufficient cell material has been formed the promoter is "switched 5 over", for example by changing the carbon source, and the number of copies of expression plasmids is increased dramatically. At the same time, the desired protein is produced in a much greater concentration; the toxicity of the protein is unimportant 10 at this stage. Surprisingly the vectors according to the invention can be used under nonselective cultivation-conditions; this is an important advantage particularly when 15 used in recombinant DNA technology processes to produce proteins. The following strains and plasmids were deposited 1 at "Deutsche Sammlung von Mikroorganismen (DSM)' 20 Grisebachstrage 8, D-3400 Gottingen on December 25 28, 1984: a) E. coli strain RRl (Identification reference RRl): DSM 3178, b) Saccharomyces cerevisiae strain SHU 32 (Identification reference SHU 32): DSM 3182, c) the plasmid: pBC3Tl transformed in E. coli 30 RRl (Identification reference pBC3Tl): DSM 3180, d) the plasmid: pADH2Bs transformed in E. coli RRl (Identification reference pADH2Bs): DSM 3179, 35 e) the plasmid: pJDB207 transformed in E. coli RRl (Identification reference pJDB207: DSM 3181. 2 14 42 - 23 - BIBLIOGRAPHY 1. Gunge, N. (1983) Ann. Rev. Microbiol. 32, 2532-2576 2. Hinnen, A. H. et al. ( 1978) Proc . Natl. Acad . Sci . USA 7_5 , 1929-1933 . 3. Struhl, K. et al. (1979) Proc .Natl .Acad .Sci .USA 7j>_, 1035-1039 4. Stinchcomb, D.T. et al. (1979) Nature 282, 39-43 5. Hsiao, C.L. and Carbon, J. (1979) Proc.Natl.Acad.Sci.USA 76, 3829-3833 6. Chan, C.S.M. and Tye, B.K. (1980) Proc.Natl.Acad.Sci.USA 77, 6329-6333 7. Hyman, B.D. et al. (1982) Proc .Natl. Acad. Sci . USA 19_, 1578-1582 8. Zakian, V. A. (1981) Proc.Natl.AcadSci.USA 78, 3128-2132 9. Fitzgerald-Hayes, M. et al. (1982) Cell 2£, 235-244 10. Zakian, V. A. and Kupfer, 11. Clarke, L. and Carbon, J. (1980) Nature 287, 504-509 12. Stinchcomb, D.T. et al. (1982) J. Mol.Biol. 158, 157-179 13. Broach, J. R. (1981) in the Molecular Biology of Yeast Saccharomyces: Life Cycle and Inheritance, J. Strathern, E. Jones and J.R. Broach, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp 445-470 14. Jayaram, M. et al. (1983) Cell _34, 95-104 15. Kikuchi, Y. (1983) Cell _35, 487-493 16. Beggs, J. (1978) Nature 275, 104-109 17. Panzeri, L. Et al. (1983) in the Abstracts of papers presented at The Meeting the Molecular Biology of Yeast, Cold Spring Harbor Laboratory, p. 69. 18. Young, T. et al. (1982) in the Genetic Engineering of Microorganisms for Chemicals, A. Hollaenaer, R.D. DeMoss, S. Kaplan, J. Konisky, D. Savage and R.S. Wolfe, eds. (Plenum Publishing , _ _ ' ' . - : •• ~ 7 . * " •' •• . ' ^ ^^ _ % [ , 2 14422 BIBILOGRAPHY (Cont) Corporation) pp. 335-36 1 19. Beier, D.R. and Young, T. (1982) Nature 300, 724-728 20. Clarke, L. and Carbon, J. (1976) Cell 91-99 21. Peacock, S.L. et al. (1981) Bioch. Biophys. Acta 6 55, 243-250 22. Birnboim, H.C. and Doly, J. (1979) Nucleic Acids Res. 7_, 1513-1523 .23. Southern, E. (1975) J.Mol.Biol. 98, 503-517 24. Sledziewski, A. and Young, T. (1982) Proc.Natl.Acad.Sci.USA 79, 253-256 25. Montgomery, D.L. et al. (1978) Cell J_4, 673-680 26. Wallace, R.B. et al. (1981) Nucl.Acids Res. 9_, 879-894 27. Miller, J.H. (1972) in Experiments in Molecular Genetics, pp. 431-433, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 28. Williamson, V.M. et al. (1981) Cell 23, 605-614 29. Bolivar, F. et al. (1977) Gene 2, 95-113 30. Tschumper, G. and Carbon, J. (19 80) Gene H)' 15 7-166 31. Chinault, A.C. and Carbon, J. (1979) Gene _5, 111-126 32. Russel, D.W. et al. (1983) J.Biol.Chem.258, 2674-2682 33. Chaconas, G. and van de Sande, J.H. (1980) Methods Enzymol. 65, 75-79 34. Twigg, A. and Sherratt, D. (1980) Nature 283, 216-218 i </div>

Claims

1* % O O } i 35 WHAT +/WE CLAIM IS:- - 25 -Claimsr 2 14422 1. A recombinant stable multi-copy yeast vector, wherein said vector contains a stabilising function 5 and a regulatable promoter by means of which the stabilising function can be controlled.

2. A recombinant stable yeast vector as claimed in claim 1, wherein the stabilising function is 10 a yeast centromere.

3. A recombinant stable yeast vector as claimed in claim 1 or 2, wherein the stabilising function is the yeast centromere 3. 15

4. A recombinant stable yeast vector as claimed in one of the preceding claims, wherein the regulatable promoter is the alcohol dehydrogenase-II promoter. 20 5. A recombinant stable multi-copy yeast vector according to one of the preceding claims further comprising an amplification system. 6. A recombinant stable yeast vector as claimed 25 in claim 5, wherein the amplification system is that of the 2-micron circle plasmid. 7. A recombinant stable yeast vector as claimed in claim 5 or 6, wherein the amplification system 30 originates from the plasmid pJDB207. 8. A recombinant stable yeast vector as claimed in one of the claims 3 to 7 wherein the yeast centromere 3 originates from the plasmid pBC3Tl. 9. A recombinant stable yeast vector as claimed in any one of the preceding claims, comprising * - 26 - 2 1 4422 as regulatable promoter ADH2 originating from the plasmid pADH2BS. 10. The recombinant stable multi-copy yeast vector 5 YCRp2. 11. The recombinant stable multi-copy yeast vector YCRp3. 10 12. The recombinant stable multi-copy yeast vector YCRp4. 13. A recombinant stable multi-copy yeast vector according to claim 1, substantially as described herein. 15 14. A process for preparing a recombinant stable multi-copy yeast vector as claimed in claim 1, which comprises inserting a regulatable promoter into a vector which has a stabilising function 20 in such a way that the stabilising function can be controlled by the promoter. 15. A process according to claim 14 wherein the stabilising function and/or promoter are as defined 25 in any of claims 2 to 9. 16. A process for preparing plasmid YCRp2 as claimed in claim 10 wherein: 30 a) the plasmid pBC3Tl is cut with BamHI, the resulting linear DNA is purified and dephosphorylated, b) the plasmid ADH2BS is cut with EcoRV, 35 the linear DNA obtained is purified and linked to a phosphorylated Bglll linker, the fragment obtained is cut with BamHI and Bglll and the about 2 14 4 k 27 1300 bp fragment purified, and c) the fragments from steps a) and b) are linked with a DNA ligase. 5 17. A process for preparing plasmids YCRp3 and YCRp4 as claimed in claims 11 and 12, wherein a) the plasmid YCRp2 is cut with Bglll and 10 BamHI and the 4.2 kb fragment is purified, b) the plasmid pJDB207 is cut with BamHI, the linear DNA obtained is purified and dephosphorylated and 15 c) the fragments from steps a) and b) are linked with a DNA ligase. 18. A process according to claim 14, substantially 20 as described herein and as illustrated with reference to the accompanying drawings. 19. A process for multiplying a heterologous DNA coding for a desired heterologous protein, 25 wherein: a) a heterologous DNA coding for a desired protein is inserted in suitable manner into a vector according to any one of claims 1 to 13, 30 b) the vector is transformed into a suitable yeast host organism which contains wild-type copies of the 2-micron circle plasmid, 35 c) the host is initially cultivated on a medium which represses the regulatable promoter, 2 1442 - 28 - d) then the host is cultivated on a medium which derepresses the regulatable promoter, e) the vectors are isolated from the cells 5 and purified and f) the heterologous DNA is isolated from the vectors and purified. 10 20. A process according to claim 19, wherein between steps (d) and (e) the host is again transferred to a medium which represses the regulatable promoter and is cultivated. 15 21. A process accordingf to claim 19 or 20 wherein the regulatable promoter is the alcohol dehydrogenase-II promoter and wherein the medium which represses the regulatable promoter contains glucose as its sole carbon source whilst the medium which derepresses 20 it contains ethanol as its sole carbon source. 22. A process for using a yeast vector as claimed in any one of claims 1 to 13 for expressing a heterologous protein, wherein: 25 a) a heterologous DNA coding for the desired protein is inserted with an expression system in correct orientation into a vector as claimed in any one of claims 1 to 13, 30 b) the vector is transformed into a suitable yeast host organism which contains wild-type copies of the 2-micron circle plasmid, 35 c) the host is initially cultivated on a medium which represses the regulatable promoter, "4 - 29 - 2 1442 2 d) then the host is cultivated on a medium which derepresses the regulatable promoter, e) and then the host is again transferred 5 to the medium which deactivates the regulatable promoter and is cultivated and f) the desired protein is isolated and purified in conventional manner. 10 23. A process according to claim 22 for using a yeast vector as claimed in claim 4 wherein said :w medium which represses the regulatable promoter contains glucose as its sole carbon source whilst 15 said medium which derepresses it contains ethanol as the sole carbon source. 24. A process for multiplying a vector according to any one of claims 1 to 13, wherein: 20 a) the vector is transformed into a suitable yeast host organism which contains wild-type copies of the 2-micron circle plasmid, 25 b) the host is initially cultivated on a medium which represses the regulatable promoter, G c) then the host is cultivated on a medium which derepresses the regulatable promoter and 30 d) subsequently the host is again transferred to the medium which represses the regulatable promoter and is cultivated. 35 25. A process according to claim 24 for multiplying the vector as claimed in claim 4 wherein said medium which represses the regulatable promoter contains i. 5 V * W - 30 - 2V4-42 2 glucose as the sole carbon source whilst said medium which derepresses it contains ethanol as the sole carbon source. 5 26. A process according to claim 19, 22 or 24, substantially as described herein. 27. A method for modifying the phenotype of a microorganism which comprises inserting into said 10 microorganism a plasmid as claimed in any one of claims 1 to 13. 28. A method according to claim 27 which comprises the steps of: 15 a) modifying the cell wall of said microorganism by treating with calcium ions; b) mixing competent cells from (a) with ligated 20 DNA containing the plasmid; and c) incubating the mixture. 29. A microorganism transformed by a plasmid 25 as claimed in any one of claims 1 to 13. 30. A yeast cell transformed by a plasmid as claimed in any one of claims 1 to 13. 30 31. An E. coli bacterium transformed by a plasmid as claimed in any one of claims 1 to 13. 32. The E. coli strain deposited as DSM 3180 and mutants thereof. 35 33. The E. coli strain deposited as DSM 3179 and mutants thereof. P 2 14422 - 31 - 34. The E. coli strain deposited as DSM 3181 and mutants thereof. 35. A method for over-producing proteins which 5 comprises cultivating a transformed microorganism as claimed in any of claims 29 to 34. -J 36. A method of claim 27 or 35, substantially as described herein. N JBi/£AREY ATTORNEYS FOR THE APPLICANTS o VEW: FH3DECB85'-./ Vv ya-'; fet </div>