JPH0158948B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to microorganisms. Specifically, it relates to a novel mutant microorganism with low protease activity. (Structure of the Invention) Yeast has cell structures and functions that are characteristic of higher organisms, and can also be used as a raw material for foods, medicines, feeds, etc.
They are useful microorganisms that are closely related to human daily life, and are expected to be developed as hosts for genetic engineering. However, when producing a heterologous protein using genetic recombination technology using yeast as a host, a portion of the produced protein is degraded by proteases, and the actually expressed protein changes, resulting in a complete target product. may not be obtained or the yield of the target product may be significantly reduced. The present inventors used the yeast Saccharomyces cerevisiae as a host to produce proteins using genetic recombination technology.
This was achieved as a result of research aimed at obtaining a host in which the produced protein is less degraded. To explain the present invention in detail, the mutant microorganism Saccharomyces cerevisiae XMF11-44 of the present invention has been deposited as FERM P-8600, and for example, the following (1) and (2) (1) Preparation of Satucharomyces cerevisiae XMF1-4 Prepared by the process of (1) Satucharomyces cerevisiae
20B-12 (α, pep4-3 , trp 1) from the well-known Satucharomyces cerevisiae S144 (a, met3 ,
leu2, gal1 , gal2 ) and Saccharomyces cerevisiae XMF1-4 (a, trp1 , pep4-
3) Select and prepare. That is, the above-mentioned 20B-12 strain and S144 strain are mixed and cultured on an appropriate medium, only diploid (zygote) cells are selectively collected, and these are grown using a liquid medium. After collection, Culture in sporulation medium.
The generated conidia are separated using a micromanipulator, and the genotype (mating type, auxotrophy, pep-4 mutation) of each spore is analyzed using standard methods, and XMF1 with the desired genotype is isolated. −
4 (a, trp1 , pep4-3 ) strain. In addition, the above 20B-12 strain and S144 strain are both
These were distributed by the YEAST GENETIC STOCK CENTER (abbreviated as YGSC, located at the University of California, Berkeley), and are included in the catalog published by the center, 5th edition (1984), page 30, and
It is described on page 21. (2) Satucharomyces cerevisiae XMF11−44
Preparation of (α, trp1 , pep4-3 , his4C , ade , ski5 ) from the well-known S. cerevisiae UK35
(α, ade , his4C , ski5 ) [Current Genetics7
Volume 449-456 (1983), published by McGill University.
obtained from Professor Howard Bussey] and the above XMF1-4 (a, trp1 , pep4-3 )
and conjugate using the same method as in (1) above, and separate the spores. Analysis of the genotype of each spore is based on mating type (α, a), auxotrophy ( trp1 ,
ade, his4C ), pep-4 mutations were carried out using standard methods, and ski5 mutations (super killer mutations) were carried out using Saccharomyces italicus IFO 0253 as a double-stranded RNA killer susceptible strain. It was tested based on the size of By the above method, XMF11-44 (α, trp1 , pep4-3 , his4C ,
ade, ski5 ) are obtained. (Effect of the invention) The microorganism of the present invention, Satucharomyces cerevisiae
XMF11-44 lacks a vacuolar protease derived from the PEP4 gene, and an extracellular phenyl methylsulfonyl fluoride (PMSF)-sensitive protease derived from the SKI5 gene. Based on the fact that S. cerevisiae
YNN27, Satucharomyces cerevisiae 20B−
12. Compared to similar yeast cells such as Satucharomyces cerevisiae S144, Satucharomyces cerevisiae XMF1-4, and Satucharomyces cerevisiae UK35, the protein produced is less likely to be degraded by protease, and the target protein can be produced in high yield. be able to. (Example) The present invention will now be described in more detail with reference to Examples. Example (1) Preparation of Satucharomyces cerevisiae XMF1-4 YEPD solid medium (yeast extract 10g, glucose
20 g, Bacto peptone 20 g and 1000 ml water) on Satucharomyces cerevisiae.
20B-12 (α, pep4-3 , trp1 ) and Satucharomyces cerevisiae S144 (a, met3 , leu2 ,
gal1, gal2 ) were mixed and held at 30°C for 30 minutes to join. This was then transferred to SD medium [0.67% yeast nitrogen base (minified amino acids), 2
% glucose] and cultured at 30°C, and only diploids (zygotes) were selectively collected. Diploid cells were grown by culturing on YEPD liquid medium, suspended in sporulation medium (consisting of 1% potassium acetate, 0.1% yeast extract, 0.05% glucose) after harvesting, and incubated at 30°C for 3-4 hours. Cultured for 1 day. Next, the generated conidia are separated using a micromanipulator, and the genotype (mating type, auxotrophy, pep4 mutation) of each spore is determined.
is analyzed using conventional methods to obtain the desired genotype.
XMF1-4 (a, trp1 , pep4-3 ) strain was obtained. (2) Satucharomyces cerevisiae XMF11−44
Preparation of (α, trp1 , pep4-3 , his4C , ade , ski5 ) XMF1-4 obtained above (a, trp1 , pep4-3 )
Strains and Saccharomyces cerevisiae UK35 (α,
ade, his4C , ski5 ) in exactly the same manner as in (1) above, and the spores were separated. The genotype of each spore was analyzed and XMF11-44 (α, trp1 ,
pep4-3, his4C , ade , ski5 ) strains were obtained. (3) Protein production using the XMF11-44 strain as a host Insert the human β-endorphin (βE) gene into an appropriate vector, and use this to
The transformed strain XMF11-44-βE-1 obtained by transforming the XMF11-44 strain was cultured, and the yield of secreted β-endorphin was measured. In the following examples, pRE059, which will be described later, was used as a plasmid into which the β-endorphin gene (βE DNA) was inserted. (b) Preparation of transformed strain Satucharomyces cerevisiae XMF11−
44 strains of cultured bacteria were collected and washed 2x.
Add TE ^buffer [10 m
M Tris-HCl (PH8.0) and 1mM
EDTA] and its 500μ
500μ of 0.2M lithium acetate was added and kept at 30°C for 1 hour. After that, 100μ of the above suspension was collected and cooled on ice, and plasmid pRE059 was added thereto.
After being kept in ice water for 30 minutes, 100μ of TE buffer containing 70% polyethylene glycol 4000 was added and mixed, and the mixture was kept at 30°C for 1 hour. Next, bacteria were collected by holding at 42°C for 5 minutes, washed with water, suspended in sterile water, applied on a selective medium, and cultured at 30°C to transform the transformed strain XMF11-
44-βE-1 strain was prepared. (b) Preparation of β-endorphin [Medium composition] Yeast nitrogen base (contains ammonium sulfate) 0.67g Dextrose 2g Water 80ml Amino acids, nucleic acids, 20ml Base mixture (*) (*) Adenine sulfate 12mg, uracil 12mg, L
-Histidine-HC112mg, L-arginine-HC112mg, L-methionine 12mg, L-tyrosine 18mg, L-leucine 18mg, L-isoleucine 18mg, L-lysine 18mg, L-phenylalanine 30mg, L-glutamic acid 60mg, L-
Aspartic acid 60mg, L-valine 90mg, L-
Threonine 120mg, L-serine 225mg, water 120mg
ml. [Culture method] Using 25 ml of the medium with the above composition, the above transformed strain was cultured at 37°C for 48 hours at a cell density (10 ⁷ cells/ml). As a result, the yield of β-endorphin was 68.8 ng/10 ⁷ cells. Ta. The number of cells is A ₆₅₀ (A ₆₅₀ = 1×10 ⁷ cells/
ml). In addition, the amount of β-endorphin can be measured using β-endorphin [RIA] Kit New England Nuclear Co., Ltd.
Radioimmunoassay was performed using catalog number NEK-003 according to the method described in the catalog. For reference, the well-known Saccharomyces cerevisiae YNN27 [protease present in the vacuole (PEP4 product) and extracellular phenyl methylsulfonyl fluoride (PMSF)-sensitive protease (SKI5 product)]
The yield of β-endorphin of the YNN27-βE-1 strain obtained by transforming the YNN27-βE-1 strain using pREI059 as a host was 47.6 ng/10 ⁷ cells. Plasmid pRE059 used in this example has the human β-endorphin precursor gene inserted between the promoter and leader sequence-containing region of the α-pheromone gene (MFα1) of Satucharomyces cerevisiae, and other markers as well. TRP1 gene,
β-lactamase gene ( ^Apr ) of pBR322, and
E.coli origin of replication (ori) and 2μm plasmid
It is a shuttle vector including ARS etc., and its details can be found, for example, in Japanese Patent Application No. 60-112287 (Japanese Unexamined Patent Publication No.
-271988), the outline of its construction is shown below. [Construction of secretory vector plasmid pRE059 containing β-endorphin gene] (1) Satucharomyces cerevisiae α factor DNA
Preparation of plasmid pLS01 (4.4 kb) containing 500 μg of chromosomal DNA of Satucharomyces cerevisiae (IFO1136) in 500 μ of buffer [100 m
M Tris-HCl (PH7.5), 50mM NaCl, 10
15 units of EcoR in [mM magnesium chloride and 1 mM dithiothreitol].
The DNA was treated overnight at 37°C, cut, concentrated, and subjected to sucrose density gradient centrifugation to collect DNA fragments of approximately 2 kb. This was added to plasmid pUC13 (Pharmacia
company catalog, P-L Biochemicals, 27 pages,
27-4973) fragment cut with EcoR
1 μg was ligated using 2 units of T4 ligase, and the resulting plasmid was used to transform Escherichia coli strain JM83, and an ampicillin-resistant ( ^Apr ) strain was selected. synthetic oligonucleotide
Colony hybridization was performed using 5'GGCCAACCAATGTACT3' as a probe, positive colonies were selected, plasmid DNA was isolated, and analysis using restriction enzymes and nucleotide sequence determination revealed the results of Cell, vol. 30, p. 937 (1982). Plasmid pLS01 containing yeast alpha factor DNA with a base sequence consistent with the description
(4.4kb) was selected. (2) Preparation of plasmid pREI032 (5.8kb) Plasmid YRp7 [5.7kb, plasmid obtained by ligating pBR322 with a fragment containing yeast TRP1,
Nature, Vol. 282, pp. 39-43 (1974)]
After partial cutting with EcoR and filling with sticky ends,
One EcoR of YRp7 ligated with T4 ligase
pREI032 (5.8kb) with the site removed was prepared. (3) Preparation of plasmid pUC8-βE (2.9kb) By cleaving plasmid pYT3-24 (described in JP-A-58-92696, p. 2) with Hae, 160bp of Hae containing βE DNA (93bp) was generated. ~
Hae fragments were collected. On the other hand, Fuage M13mp7 (Nucleic Acids Re
-RFDNA of search volume 9, pages 309-321)
(double-stranded DNA) is cut with Hinc, and ethanol is added to dissolve the fragment in ethanol.
After removing 5′GACCTGCAGGTC3′ (Hinc to Hinc), the 160 bp Hae to Hae fragment containing the βE DNA was ligated to the Hinc site using T4 ligase. Introducing this into E. coli HB101 strain,
RFDNA was prepared from the obtained transformed strain and cleaved with BamH.
The ~BamH fragment was added to plasmid pUC8 [Bethesda
BRL catalog published by Research Laboratories, Inc. (August 1, 1983), Cat/No. 53595A]
pUC8−βE by inserting it into the BamH site of
(2.9kb) was obtained. (4) Preparation of plasmid pREI046 (7.4 kb) (a) pLS01 (4.4 kb) was cut with EcoR and Hind to obtain an EcoR-Hind fragment (1.4 kb) containing the promoter sequence and leader sequence. (b) pUC8−βE (2.9kb) by Hind and EcoR
Hind~EcoR fragment (0.2kb)
I got it. (c) pREI032 (5.8 kb) was cut with EcoR and ligated with the DNA fragments obtained in (a) and (b) above using T4 ligase to obtain pREI046 (7.4 kb) (Figure 1). ). (5) Preparation of plasmid pREI052 (5.2 kb) (a) pREI032 (5.8 kb) was cut with EcoR and Pst to obtain an EcoR-Pst fragment (0.8 kb) containing TRP1. (b) Cut the 2μm plasmid with EcoR, fill in the sticky ends to make blunt ends, and then insert Pst
Pst~EcoR containing the replication origin by cutting with
A fragment (2kb) was obtained. (c) The DNA fragments obtained in (a) and (b) above,
Pvu obtained by cutting pBR322 with EcoR and Pvu
〜Create pREI051 (5.1 kb) by ligating with EcoR fragment (2.3 kb) using T4 ligase, partially cutting with EcoR, filling in the sticky ends and ligating with T4 ligase to make blunt ends, and cut one EcoR cleavage site. was deleted
pREI052 (5.2kb) was obtained (Figure 2). (6) Preparation of plasmid pREI059 (6.8kb) (a) Cut pREI046 (7.4kb) with EcoR and Sma to generate EcoR~ containing the promoter sequence, leader sequence, and βE DNA sequence.
Sma fragment (1.6kb) was collected. (b) Hinc containing the terminator sequence by cutting pLS01 (4.4kb) with Hinc and EcoR
EcoR fragment (0.3kb) was collected. (c) After cleaving pREI052 (5.2kb) with EcoR and adding bacterial alkaline phosphatase (BAP) to remove the terminal phosphate group, the DNA fragment obtained in (a) and (b) above and ligated using T4 ligase plasmid pREI059
(6.8kb) was obtained (Figure 3).

[Brief explanation of drawings]

Figures 1 to 3 are plasmids used in the present invention.
This is a schematic diagram showing the configuration route of pREI059, in which E stands for EcoR, H stands for Hind, and S stands for Sal.
, P is Pst, B is BamH, Pv is Pvu
, Sm is Sma, Hc is Hinc, X is Xba
are shown respectively.

Claims (1)

[Claims] 1 Satucharomyces cerevisiae 20B-12,
Satucharomyces cerevisiae XMF1− obtained by mating with Satucharomyces cerevisiae S144
4 and Satucharomyces cerevisiae UK35, and which has lower protease activity than similar yeast strains, Satucharomyces cerevisiae XMF11-44 (Feikoken Bibori No. 8600).