JP2850262B2 - How to maintain a stable plasmid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for stably retaining a recombinant plasmid in yeast when producing a heterologous protein using genetically modified yeast.

Conventional technology When a biologically active substance is produced using gene recombination technology, the stable maintenance of the recombinant plasmid in the host cells is important for maintaining the high productivity and for the production of the substance. It is also essential to guarantee quality. Various attempts have been made to stably maintain a plasmid even in a recombinant using yeast as a host. One of them is a method of integrating a target gene into a chromosome. However, in this method, the number of copies of a target product-producing gene per cell is small, and sufficient productivity cannot always be obtained. A plasmid vector having a replication origin of 2 μm plasmid is 2 μm
Compared with a yeast having no plasmid (strain cir ⁰ ), it is more stably maintained in a yeast having a 2 μm plasmid (cir ⁺ strain), but there is a problem that the proportion occupied by the vector differs significantly from clone to clone. Plasmids with a chromosomal origin of replication ARS have high copy number but poor stability in the host. In order to solve this point, a replication start point of 2 μm and AR
An expression plasmid with S has also been developed.

Problems to be Solved by the Invention However, conventional knowledge alone cannot always stably maintain an expression plasmid for a target gene in a host yeast, and further development of a new technology is essential.

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted various studies on substances capable of stably retaining a plasmid in yeast cells by adding the medium, and as a result, histidine was added. It has been discovered that the plasmid can be stably maintained by culturing the recombinant in the obtained medium, and as a result of various studies, the present invention has been completed.

That is, the present invention provides a method for stably maintaining a recombinant plasmid in yeast cells, comprising culturing a yeast holding the recombinant plasmid in a medium to which 150 mg / l or more histidine has been added. Things.

As the recombinant plasmid used in the present invention, YRp
And a plasmid capable of replicating in yeast having a vector of the type or YEp type or both. More specifically, a modified hepatitis B virus surface antigen (HBsAg) P31 protein yeast expression plasmid pGLD P31-RcT (JP-A-63-109795) and a human lysozyme yeast extracellular expression plasmid pGEL
125 (JP-A-64-10986) and the like are advantageously used.

As the host used in the present invention, Saccharomyces
Cerevisiae (Saccharomyces cerevisiae), more specifically Saccharomyces cerevisiae AH22R ^- (leu2 his4 ca
nl cir ⁺ pho80) [Proc.Ntl.Acad.Sci.USA, 80 , 1 (198
3)] or a derivative thereof and the like, and a known method [H
innen, A, et al., Proc. Natl. Acad. Sci. USA, 75 , 1927 (197
8)] or a recombinant plasmid is introduced by a method analogous thereto.

When culturing the yeast recombinant thus obtained, a medium to which histidine is added is used. As histidine, L-histidine or DL-histidine and their contents are used. The added amount is 150 mg / l to 1000 mg / l as L-histidine, more preferably 200 mg / l.
Or 500 mg / l. The above amount of histidine is added to the medium when culturing a yeast recombinant to produce the target substance encoded by the gene in the recombinant plasmid and during culturing for subculture. Prior to culture for producing the target substance, preculture for propagation of the yeast recombinant may be performed. The amount of histidine to be added to the medium used for the preculture may be smaller than the above-mentioned amount (for example, 100 mg / l to 150 mg / l as L-histidine).
Degree), preferably the above amount (150 mg / l to 1000 mg /
(preferably 200 mg / l to 500 mg / l) of L-histidine is also used for the preculture. As the medium, a liquid or agar medium known per se is used,
For example, Barkholder's minimal medium [Bulkholder.P.
R.:Am.J.Bot. 30 , 206 (1943)] and Bacto East Nitrogen Base (Difco). If the recombinant shows auxotrophy, the required product is added as needed. In some cases, a small amount of a natural product such as casamino acid, yeast extract or malt extract can be added.

Culture of yeast recombinants is usually 15 ° C to 40 ° C, preferably
The reaction is carried out at 20 to 35 ° C. for 1 to 5 days, preferably 2 to 4 days, and if necessary, aeration and stirring can be added.

When the target substance is secreted out of the yeast recombinant cells,
After the culture, the culture supernatant can be subjected to various known separation and purification methods to isolate and purify the target substance. If the target substance is not secreted outside the cells of the yeast recombinant, the cells are collected after culturing, and then lysed with Zymolyase [manufactured by Kirin Brewery Co., Ltd.], or subjected to sonication or glass beads. After the cells are destroyed by mechanical disruption and the target substance is extracted, the extract can be subjected to various separation and purification methods known per se to isolate and purify the target substance. The obtained purified product can be used according to the purpose (for example, treatment or prevention of a human disease) in the same manner as a sample produced by a conventional method.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Saccharomyces cerevi
^{siae) AH22R - / pGLD P31-} RcT the Institute for Fermentation (IF
O) was deposited as IFO 10206, and FERM BP-105 was registered with the Research Institute of Microbial Technology (FRI) of the Ministry of International Trade and Industry of Japan.
No. 9 has been deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in Patent Procedures (Budapest Treaty).

Saccharomyces cerevi
siae) AH22R ^- / pGEL125 was deposited as IFO 10211 in IFO, also has been deposited under the Budapest Treaty as FERM BP-1345 to FRI.

Example 1 Modified HBsAg P31 protein producing recombinant Saccharomyces
Cerevisiae AH22R ^- / pGLD P31-RcT the 2l Erlenmeyer before were charged to the flask culture medium 1 (1 per glucose 100g,
Potassium monohydrogen phosphate 3g, L-asparagine 4g, L-histidine 100mg, magnesium sulfate 500mg, calcium chloride 330m
g, potassium iodide 0.1mg, ferrous sulfate 2.5mg, copper sulfate 0.4mg,
0.4 mg manganese sulfate, 0.2 m ammonium phosphomolybdate
g, zinc sulfate 3.1 mg, inositol 30 mg, thiamine 0.6 mg, pyridoxine 0.6 mg, calcium pantothenate 0.6 mg, niacin 0.6 mg, biotin 0.006 mg), incubate at 30 ° C for 3 days, and preculture 2.5 liters of a production medium [200 g of a sucrose, 2.5 g of sodium monohydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, each containing 200 ml of a liquid in a 5 liter fermenter]
Ammonium sulfate 2g, magnesium sulfate 0.5g, calcium chloride 0.33g, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate
10mg, potassium iodide 0.1mg, inositol 90mg, thiamine hydrochloride 1.8mg, pyridoxine hydrochloride 1.8mg, nicotinic acid 1.8mg, calcium pantothenate 1.8mg, biotin 0.018mg]
In addition, L-histidine was 100 mg / l, 150 mg / l, 200 mg / l, 400 mg / l.
The cells were transplanted to each of the added culture mediums so as to obtain 1 l, and cultured under aeration and stirring at 30 ° C. for 3 days. The cultured solution was plated on a plate medium [yeast-nitrogen base (amino acid free): 6.7 g, glucose: 20 g, L-histidine: 100 mg, adenine: 30 mg, uracil: 30 mg, agar: 200 g]. The productivity of the modified HBsAg P31 was examined, and the results shown in Table 1 were obtained.

Example 2 The L-histidine concentration of the preculture medium of Example 1 was 100 mg /
l (A medium) and 400 mg / l 2 kinds of media were prepared with the (B medium), Saccharomyces cerevisiae AH22R ^- / pGLD P31-
After inoculating RcT and culturing at 30 ° C. for 3 days, subculture was repeated in each medium for 3 days, and the productivity of the modified HBsAg P31 was examined. The results in Table 2 were obtained. That is, when the cells were passaged in the A medium, the productivity was significantly reduced, but when the cells were passaged in the B medium, they were completely stable after the fifth passage.

Example 3 The L-histidine concentration of the preculture medium of Example 1 was 100 mg /
l (A medium) and 400 mg / l (B medium)
Prepare one by one in a 2 l flask, and use L-
The histidine concentration was 100 mg / l (A 'medium) and 400 mg / l (B'
Medium) in a 50-liter fermenter.

Human lysozyme gene expression plasmid by recombinant Saccharomyces cerevisiae AH22R ^- / pGEL125 strain preculture medium (A, B medium) was inoculated and cultured for 3 days at 30 ° C., the culture in A medium production medium A 'medium The obtained culture was transplanted to the B 'medium, and the culture was cultured in the B medium. 50
l After culturing for 4 days in a fermenter under aeration and agitation conditions, the culture solution was diluted with physiological saline to determine the expression of human lysozyme agar medium [per sucrose 80 g, L-asparagine 5 g, potassium chloride
2 g, L-histidine 300 mg, potassium iodide 100 μg, potassium dihydrogen phosphate 400 mg, magnesium sulfate 500 mg, calcium chloride 330 mg, ferrous sulfate 1.25 mg, copper sulfate 0.2 gm, manganese sulfate 0.2 mg, ammonium phosphomolybdate 0.1 mg , Zinc sulfate 1.55mg, inositol 20mg, thiamine 0.4mg, pyridoxine 0.4mg, calcium pantothenate 0.4mg, niacin 0.4m
g, biotin 0.004 mg, glucose 10 g, 1 M-tris-maleic acid (pH 6.5) 25 ml, Micrococcus rhizodyticus 5 g, agar 20 g], and cultured at 30 ° C. for 5 days.
Depending on the presence or absence of plaque around the growing colony,
The productivity of human lysozyme was examined, and the results in Table 3 were obtained.

According to the present invention, according to the present invention, a recombinant plasmid which is likely to be generated during the cultivation of a yeast recombinant is dropped out of the host yeast and a part of the gene is removed from the recombinant plasmid by self-recombination of the recombinant plasmid. Dropout can be suppressed, and the target substance can be stably produced.

The method of the present invention is advantageously used for culturing to produce a useful substance of interest and subculture of yeast recombinants.

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Claims (1)

(57) [Claims] (1) A yeast containing a recombinant plasmid is
A method for stably maintaining a recombinant plasmid in a host yeast, comprising culturing in a medium supplemented with histidine of not less than g / l.