JP2694210B2 - Yeast expression vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a yeast expression vector, more specifically, the following restriction enzyme map, It is surrounded by each cleavage site of Sal I and Xba I in the 3'side peripheral region of the yeast GAL7 gene as a transcription termination sequence in
It relates to a yeast expression vector containing 8 base pairs. [Prior Art] Conventionally, PGK (phosphoglycerophosphate kinase), GPD (glyceraldehyde-3-phosphate dehydrogenase), PHO5 (inhibitory acid phosphatase) have been used as expression vectors utilizing a yeast controllable promoter. What is used and GAL genes (galactose metabolism system, hereafter GAL
, For example, GAL1 , GAL10 , GAL7, etc. are known) and a series of those using. [Problems to be Solved by the Invention] In these expression vectors, the GAL system is superior in terms of easiness of control technology. Among these, expression using the control region of the GAL7 gene is preferable. Even if a vector is used to express a foreign gene, that is, to produce a protein, it has been previously reported in terms of transcriptional expression.
Transcription does not terminate at the GAL7 transcription termination point, and thus the production efficiency of the produced protein was poor. Further, no one has attempted to increase the production efficiency by using the transcription termination sequence of GAL7 (hereinafter referred to as terminator) or the positive regulatory gene GAL4 . Furthermore, the expression efficiency is said to differ significantly with and without a terminator [R. Derynck et al., Nucleic Acid
s Res., 11: 1819 (1983)], a vector having a cloning site between a promoter and a terminator was considered to have good expression efficiency. However, the terminator was different from the promoter, and it was said that no difference in the strength of its function was observed [GABitter et al., Gene, 32:
263 (1984)]. [Means for Solving Problems] 1. The present inventors have succeeded in producing a human adenovirus E1a protein in yeast using an expression vector utilizing the control region of the GAL7 gene. (Presentation at the 8th Annual Meeting of the Molecular Biology Society)
In the downstream of ac DNA, it has been reported in the past [Tajima, Nogi, Fukasaw
a, 1985 Yeast 1 (1)], a GAL7 transcription termination point and a 411 base pair fragment upstream (including the translation region) of the GAL7 transcription termination point were inserted as a "terminator". However, subsequent studies revealed that transcription did not stop at the expected position, and most continued further downstream. We then added a 328 base pair fragment further downstream from what was traditionally believed to be the transcription termination point, and surprisingly transcription terminated completely at the expected position. Furthermore, it was surprisingly found that the production efficiency of the E1a protein also increases accordingly. Furthermore, recent studies, sequence has been to include terminator in JP 60-24818, it is not necessary for transcription termination, and actually required portion of the 328 base pair, S
It was found to be a base pair downstream of al I. 2. One of the inventors of the present invention, Fukasawa, was working with a collaborator (Hashimoto et al., 1981) on the positive regulatory gene GAL4.
It was reported that the expression level of GAL gene was increased when the clone was cloned and ligated to a multicopy vector to increase the copy number. Similar phenomena occur in Johnston and Ho
Also reported by pper. Applying the fact, it was found that when GAL4 was inserted into the GAL7 expression vector, the production efficiency of E1a protein was increased. At this time, the medium before induction with galactose, it is preferable to use those that use glucose as a carbon source, in the conventional GAL- based expression vector, suppression of expression by glucose in the medium, delay is observed. . However, it was found that the addition of GAL4 eliminated this glucose effect. The present invention has been completed based on these findings, and the following restriction map, It is surrounded by each cleavage site of Sal I and Xba I in the 3'side peripheral region of the yeast GAL7 gene as a transcription termination sequence in
The present invention relates to an expression vector containing 8 base pairs. The yeast vector used in the present invention is generally prepared as a shuttle vector between Escherichia coli and yeast like the yeast vector. The use range of the present invention is limited to any yeast expression vector as the terminator and GAL4 as a GAL- based expression vector. The origin of replication of the vector improved by the present invention in yeast may be a commonly used YEp-type plasmid (having a replication origin of yeast 2 μm DNA) or YRp-type plasmid (having an autonomously replicating sequence obtained from yeast chromosomal DNA). ) Or the like can be used. A polypeptide derived from a foreign gene is preferably a YEp type plasmid which has a large copy number per cell for production and is stably retained. Yeast selectable marker DNA is required to select for yeast cells transformed with the recombinant vector. Examples of selectable markers include cloned genes such as TRP1 , LEU2 , URA3 , and HIS3 that complement commonly used auxotrophic mutations. Of these, URA3 is particularly preferable.
In addition, an antibiotic resistance gene for selection using drug resistance can also be used. The foreign gene that can be ligated to the expression vector improved by the present invention may be a gene containing an intron or cDNA in Saccharomyces cerevisiae . For other eukaryotic genes, cDNA is essential.
In addition, since prokaryotic genes do not contain introns, they can be used as they are. In addition, artificially prepared DNA corresponding to amino acids can be preferably used. All open reading frames of the gene encoding the protein can be expressed. Regardless of the type of protein to be expressed, even if the protein is harmful to yeast growth, it can be sufficiently expressed by the present galactose-inducible expression vector. For example,
Insulin, platelet-derived growth factor (PDGF), interferon (α, β, γ), gonadotropin, growth hormone, enkephalins, endorphins, adrenocorticotropic hormone (ACTH), calcitonin, parathyroid hormone, nerve growth factor (NGF), erythropoietin, blood coagulation factor, urokinase, serum albumin, cytokine, epidermal growth factor (EGF), prolactin, rennin,
Placental lactogen, thymopoietin, gastric secretion inhibitory polypeptide (GIP), cholecystokinin (CCK-39), gastrin (BG), calcitonin, glucagon, secretin,
It can be suitably used for proteins such as motin, somatostatin, pigment cell stimulating hormone, elastin, collagen, lipase, lactase, β-galactosidase, invertase, cellulase, amylase and protease. It can also be applied to the production of viral proteins such as human adenovirus E1a protein, SV40T antigen, herpesvirus IE protein, etc. in yeast. In the present invention, the GAL7 terminator is inserted immediately downstream of any polypeptide gene that is a useful substance. Examples of the terminator of GAL7 include the restriction enzyme Sal I , Xba I fragment 328 bp fragment found by the present inventors. The Sal I , Xba I fragment 328 bp can be prepared by a conventionally known DNA fragment preparation method. Further, this DNA fragment can be inserted directly downstream of the useful substance gene by the conventionally known DNA fragment insertion method. The yeast hosting the expression vector is Saccharomyces
Cereviche ( Saccharomyces cerevisiae ) and, for example, Saccharomyces karevisbergensis ( Saccharomyce
s carlsbergensis) related species, such as is preferably used. Transformation of yeast with an expression vector is performed by the method of Hinnen et al. [Proc. Natl. Acad. Sci. USA., 75: 1929 (1978)].
And Kimura et al. [J. Bacteriol. 153: 163 (1983)]
Either method can be used. Hereinafter, embodiments of the present invention will be described in detail. In order to demonstrate the usefulness of the yeast expression vector improved by the present invention, the following recombinant vector was prepared to obtain transformed yeast, and E1a protein was expressed. That is, FIG. 1A shows that the expression vector for the E1a protein by yeast was previously prepared by a part of the present inventors and the GAL
A plasmid in which 4 is inserted, yeast 2 μm DNA as an origin of replication, URA3 as a yeast selection marker, and a regulatory gene GA
A vector (hereinafter referred to as pTW3433) into which L4 , GAL7 as a promoter, and a DNA sequence expressing the human adenovirus E1a protein as a foreign gene (hereinafter referred to as E1a sequence) were cloned. FIG. 1B and FIG. 1C show a method for preparing pTW3433. E. coli M15 containing the plasmid pTFG4H3 containing the GAL4 gene
/ pTFG4H3 has been deposited as Escherichia coil M15 (pTFG4H3) at the Institute of Biotechnology, Institute of Biotechnology. The deposit number is Microorganisms Research Institute No. 9373, FERM P-9373. FIG. 2 shows the E1a protein production by the yeast transformed with the above vector pTW3433 as an electropherogram by Western blotting. (The E1a protein expression result by the vector lacking GAL4 regulatory gene was shown as a control.) That is, at each time shown in the figure, 5 ml of the culture solution was collected, and the cells were collected by centrifugation, and then 10 mM Tris buffer (1 mM EDTA, containing 1 mM dithiothreitol). This was suspended in 0.1 ml of the same buffer (containing 1 mM phenylmethylsulfonyl fluoride), transferred to an Eppendorf tube, and then glass beads (diameter 0.5 mm)
Mix with 0.1 g and shake in a cold room on a vortex mixer for 5 minutes or more. The supernatant obtained by centrifuging this with a microfuge type centrifuge (10,000 rpm) for about 5 minutes was used for evaluation of E1a expression. Western blotting is based on Towbin, Staehhelin, Gordon
(Pro, Natil. Acad. Sci. USA. 76, 4350-4354, 197.
9 years). As is clear from FIG. 2, the presence of the GAL4 regulatory gene not only increases the expression level but also shortens the recovery time from glucose repression after the addition of the inducer galactose. FIG. 3A shows a vector into which the Sal I and Xba I fragment 328 bp containing the GAL7 terminator for showing the effect of the terminator on the expression of E1a protein by yeast was inserted, and a vector lacking the vector. Has been done. The manufacturing method is as shown in FIG. 3B. That is, pYN21 containing the terminator shown in the restriction map of Fig. 3B was transformed into Escherichia coli M15 (pYN21
N21)] has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology. The deposit numbers are Microorganisms Research Institute No. 9374, FERM
P-9374. (However, no regulatory gene GAL4 ) Origin of replication: 2 μm DNA Selectable marker: URA3 Promoter: GAL7 Terminator: Sal I , Xba I fragment 328 bp pYN21 including the terminator shown in the restriction enzyme map in Fig. 3B is the restriction enzyme map below. , It is surrounded by each cleavage site of Sal I and Xba I in the 3'side peripheral region of the yeast GAL7 gene as a transcription termination sequence in
It contains 8 base pairs. Such a plasmid pYN21
Is the GAL gene and Bgl II and Eco in the 3'peripheral region.
It is prepared by ligating a 2.1-kilobase pair fragment surrounded by the RI cleavage site to the BamHI and EcoRI sites of the known vector pBR322. FIG. 4 shows E1 produced by the expression vector of FIG. 3A.
[Fig. 3] Fig. 3 shows an analysis electropherogram of a protein by Western blotting. It can be seen that the system A without the terminator produces less E1a protein than the system B with the terminator, and therefore the time required to accumulate the same amount is delayed. FIG. 5 shows the structure of the most preferable yeast expression vector of the present invention, which has the following constitution. Origin of replication: 2 μm DNA selectable marker: URA3 regulatory gene: GAL4 promoter: GAL7 foreign gene: E1a sequence terminator: Sal I, Xba I fragment 328 bp Figure 6 shows that yeast transformed with this expression vector was used to produce E1a protein. The results are shown in an electropherogram by Western blotting. Sixth
As is clear from the figure, the combination system of the regulatory gene GAL4 and the terminator Sal I, Xba I fragment 328 bp produces a large amount of E1a protein, the initiation of the production is rapid, and the productivity in glucose medium is not observed at all. The advantages have been demonstrated. First, yeast transformed with an expression vector improved by the present invention containing a foreign gene lacks a selectable marker nutrient (uracil in the example), uses glucose as a carbon source, and has a logarithmic growth phase to a stationary phase in a medium. Let it grow. In addition, when the regulatory gene GAL4 is used in the present invention, the recovery time from so-called glucose repression is extremely shortened, so that the production of the polypeptide reaches its maximum value quickly. Next, a certain amount of this bacterium was added to glucose 0.1-0.5% (6th
1% galactose 1-5% (2% in Fig. 6)
%) Inoculating a medium lacking selective nutrients
After 12-18 hours, the maximum production of the polypeptide from the foreign gene is reached. Returning to FIG. 1A again, this FIG. 1A shows 2 μm DNA, URA
3 , the structure of plasmid pTW3433 consisting of GAL4 , GAL7 promoter and E1a gene is shown, and the construction procedure is shown in detail in FIGS. 1B and 1C. In FIG. 1B, first, E1 of the human adenovirus genome
In order to remove the intron in the a region, it was replaced with a fragment surrounded by Sma I Xba I of adenovirus type 2 cDNA. Next, after treating the 5'side with Bal 31 to remove about 560 bases,
Bam HI linker was connected. The 3'side was cleaved with Hpa I and then connected with a Hind III linker. In FIG. 1B, the blackened portion shows the E1a coding region. This E1a
The expression vector pTM3 containing the yeast GAL7 promoter
Substitutions were inserted into the BglII and HindIII regions of 4 as shown in the figure. Figure 1C shows the procedure for inserting GAL4 into pTM34 / 3E1a constructed in Figure 1B. FIG. 2 shows the results of Western blotting showing the effect of the regulatory gene GAL4 on the expression of E1a protein in yeast. First, MF8-1 strain ( a ade , his 3, leu 2, trp 1, ura 3)
A transformant obtained by introducing pTW3433 into glucose was used as a glucose carbon source, and a medium lacking uracil (SGlu: composition; 0.5% casamino acid, 0.67% Nitrogen Base W / O amino acid, 0.002
% Adenine, 0.002% tryptophan, 2% glucose). When the total number of cells reached about 1 x 10 ⁶ / ml, the cells were collected and transferred to SGal (medium added with galactose at 2% instead of glucose of SGlu). Approximately 5 ml samples were taken over time. Prepare the crude aliquot as described in the text and add 0.1% SDS.
Fractionated by% polyacrylamide electrophoresis. The fractionated proteins are electrotransferred and attached to a nylon membrane and then treated with anti-E1a rabbit serum. Next, this is treated with anti-rabbit IgG goat IgG to which peroxidase is bound, and then developed with hydrogen peroxide solution and diaminobenzidine hydrochloride. A quantitative comparison of E1a was carried out at 450 nm using a Shimadzu Double Beam Chromatoscanner CS-930. FIG. 3A shows the regulatory gene GAL4 from the expression vector of FIG.
The structures of the plasmid (B) with and without the terminator Sal I, the Xba I fragment 328 bp, and the plasmid without (A) without the terminator are shown. The construction procedure is as detailed in FIGS. 1B and 3B. FIG. 4 shows the results of analysis of the E1a protein expressed by the plasmid of FIG. 3A in an electropherogram by Western blotting. The MT8-1 strain was inserted with the plasmid A shown in FIG. 3A and the plasmid B containing the terminator inserted thereinto by transformation. SGly these yeasts
Lac (a medium containing 2% each of glycerol and lactic acid instead of glucose of SGlu in FIG. 2) is inoculated. Total number of bacteria about 1
At the time of × 10 ⁶ / ml, the time of adding galactose to 2% was set to 0, and the second time was added at each time shown in the figure.
Samples were collected, processed and analyzed by Western blotting as in the figure. FIG. 5 shows the structure of the plasmid of FIG. 3A with the regulatory gene GAL4 added. FIG. 6 shows an electrophoretogram of the E1a protein expressed by the plasmid of FIG. 5 analyzed by Western blotting. That is, the plasmid shown in FIG. 5 was transformed into MT8-1 strain, and this was precultured in SGlu until the late logarithmic growth phase. Glucose 0.1
%, Galactose 2%, was inoculated into a medium (SGluGal) so that the total number of bacteria was about 3 × 10 ⁵ / ml, and thereafter, samples were taken at each time shown in the figure. Subsequent processing was exactly the same as that shown in FIG.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows 2 μm DNA, URA3 , GAL4 , GAL7 promoter,
1 shows the structure of plasmid pTW3433 consisting of the E1a gene. FIG. 1B and FIG. 1C show the procedure for constructing the plasmid pTW3433. FIG. 2 shows the results of Western blotting analysis showing the effect of the regulatory gene GAL4 on the expression of E1a protein in yeast. FIG. 3A shows the structures of the plasmid (A) lacking the regulatory gene GAL4 from the expression vector of FIG. 1A, without the terminator Sal I and Xba I fragment 328 bp, and with the plasmid (B). FIG. 3B and FIG. 1B show the procedure for preparing the plasmid of FIG. 3A. FIG. 4 shows the analysis results of the E1a protein expressed by the plasmid of FIG. 3A by Western blotting. FIG. 5 shows the structure of a plasmid in which the regulatory gene GAL4 is added to the plasmid of FIG. 3A. FIG. 6 shows an electropherogram of the E1a protein expressed by the plasmid of FIG. 5, which was analyzed by Western blotting.

Claims (1)

(57) [Claims] The restriction map below, It is surrounded by each cleavage site of Sal I and Xba I in the 3'side peripheral region of the yeast GAL7 gene as a transcription termination sequence in
A yeast expression vector comprising 8 base pairs. 2. The yeast expression vector according to claim 1, which has the sequence of the positive regulatory gene GAL4 . 3. The yeast expression vector according to claim 1, which comprises a gene encoding an arbitrary polypeptide downstream of the promoter region and upstream of the terminator region.