JPH04501662A - Mixed feeding recombinant yeast fermentation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Mixed feeding recombinant yeast fermentation The present invention relates generally to significant improvements in fermentation technology, and in particular to the use of recombinant yeast. Controlled high production with recombinant yeast host strains to improve production produced by the expression of a heterologous gene encoding a foreign protein in It allows the preparation of increased amounts of protein. Therefore, the original In fermentation at high cell concentrations to maximize recovery of foreign gene products, The yeast host used in a given recombination allows for the production of recombinant proteins. Takes advantage of major improvements in propagation and culture techniques. The present invention is particularly applicable to Pichia paths. Tris (Pichia (P.) pastoris) is suitable for use with yeast hosts. However, all yeast hosts with similar culture characteristics and susceptibilities, i.e. Hansenula polymorpha also originated from According to Akira, it is assumed that it can be used successfully.

For the production of large amounts of biologically useful heterologous gene products or heterologous polypeptides , recombinant, where the expression of a heterologous gene (relative to the yeast host) is used in large-scale fermentations. Yeast host strains have been found to be particularly amenable for use in host systems.

These yeast hosts appear to combine the advantages of both prokaryotes and eukaryotes. It also allows for easy development when setting up recombinant fermentation so that it can be explored to the fullest. This property makes it particularly suitable for recombinant systems. for example Like prokaryotes, yeast are unicellular organisms with relatively fast growth rates and Relatively easily, regulatory elements can be operably placed in a given heterologous gene to create a heterologous DNA. transformed with the cassette. Moreover, it is usually considered to be unique to higher eukaryotes. It has the advantage that it can be used to incorporate glycosylation structures into organs and other In some cases, the heterologous gene product can be more easily cultured in host yeast tissues. Such as the use of secretory pathways to allow recovery from the culture medium.

Recently, the yeast host P, Bastris has attracted much attention. This methyltrophic yeast has been shown to be a prominent yeast for high-level heterologous gene expression. Departing from Pichia The overall success of the current system is due to its primary alcohol oxidase (^0X1) gene. It is linked to the strength of the gene promoter, and the aforementioned strength is When grown in pure P, up to 30% of the protein in pastoris extracts oxidase enzymes. AOX1 gene The promoter of P and bus was isolated and cloned as early as 1985. It has been transformed into Tris. Other important characteristics of this methylotrophic yeast host i can reach high cell concentrations economically in a simple methanol-nutrient-salt medium. It means that it can be done. Under these conditions, the hepatitis B virus surface antigen high levels of heterologous gene products, including invertase, animal lysozyme C, and others. It has been shown that it is possible to produce These heterologous gene products include the product is secreted into the medium supporting the host cells and can be isolated from the medium. Some have the additional advantage of being recognized by the chia secretory process.

Reports of yeast that can use methanol for growth first appeared in the late 1960s. . Since then, only a few methylotrophic yeasts have been identified; Candida, Hansenula, Torulopsis It belongs to the 4 genus named ``psis''. Biochemistry of methanol utilization in these genera The route has been clarified and has been shown to be similar to the neck. “Methyl nutrition” The term refers to yeast's ability to grow on methanol, among other carbon source nutrients. , the researchers combined methanol and glucose mixtures simultaneously or with ginger, formic acid, This is a mixed carbon source with glycerol, ribose, sorbitol, and xylose nutrients. We found that these strains are available. Cleeson et al. , Y e a s t 4.1 (1988).

Research has focused on a specific genus of yeast called Hansenula, especially the methylotrophic species Hansenula poly This has also been done for Morpha. This study aims to develop supporting media to promote growth rates. The focus is on the study of soil mechanisms and the differences in carbon-limited continuous culture device culture systems. This includes studies of carbon utilization by species from media with different compositions. For all mixtures As a result of our research, it became clear that similar usage patterns developed; This means that at low dilutions methanol and higher carbon sources are utilized simultaneously, but at higher dilutions cells prefer higher-order carbon sources (i.e., compounds consisting of more carbon than carbon) On the other hand, unused methanol often accumulates in the culture medium. The pattern was that Egl i, Biotech nology and Bioengineering 28.1735 (1986 ) and the various references cited therein as reports of similar studies. Light.

Researchers in the same laboratory have investigated a mixture of carbon sources (C1 and C6) containing a high proportion of methanol. When using a compound, a certain multiplication factor for methanol makes methanol the only Recorded in response to the multiplication seen with methanol when used as a carbon source It had been. Additionally, the researchers found that glucose, for example, is present at high levels in the culture medium. They discovered that when present, the methanol metabolic pathway is inhibited. glucose is limited Disinhibition of the methanol pathway was observed when present at a concentration of This latter system Increasing the methanol concentration in the system increases the induction of the methanol metabolic pathway by methanol. guidance was observed. The degree of induction is determined by increasing the ratio of methanol in the mixed substrate mixture. It was found that the rate increases with the rate. Eagly^rchives of M icrobiology 132, H1982), Reference Eagly et al., Mierob ial Growth on cl Compounds, Crawford et al. (C rawford) eds, American 5ociety for Mi crobiology, Washington, dAc, (198 4); Eagly et al., Journal of General Microbio 132, 1779 (1986); Egeling et al. ng)^rchives of Microbiology 127,119 (1980); Egeling et al. hives of Microbiology 132, 362 (1981); Hazeu et al. (Hazeu) Biotech 1etters T, 399 (1983); Mueller et al., Microb, Biotech, 25, 238 (1986); Swartz et al. ^pp1. Envir, Microbio 1.41.1206 (1981).

Attention is also being focused on various future jobs such as:

All of these earlier researchers worked in continuous culture device systems, so they were not controlled. studies that caused changes in the concentration of specific nutrients in the overall culture medium. . In addition, these researchers use cultures of non-recombinant organisms and A relatively low concentration of culture medium was used. Therefore, these researchers The mother organism mutates or otherwise foreign genes and their functional production. A system that is less susceptible to the genetic burden of removing things, and a system that This is known to occur when high culture densities are used to develop a limited cell culture volume. I was experimenting with a system that had no problems due to irregularities.

Moreover, the researchers avoided other problems associated with high-density fermentation. virtue In addition, high-density fermentation often involves the provision of high substrate feedstock concentrations and/or supported by the addition of a high percentage of fees. This parameter is similar to polycarbon yeast, in that ethanol, a byproduct of substrate metabolism, can accumulate in the growth medium. is known to affect the physiological functions of Ethanol is an alcohol oxidizer. It can be caused.

In contrast, research on recombinant yeast systems has Maximize production levels of heterogeneous products to isolate and recover available quantities Such high densities require highly developed culture manipulation techniques. These host cells are , a culture that is susceptible to variations in growth patterns caused by enhanced culture modification. newcomers to their surrounding environment, as well as extreme exposure to nurturing techniques. must be able to withstand the enormous production of foreign gene products. This host's life Physical reactions eliminate or eliminate the production of foreign proteins by inducing mutations. It is predicted that the aim is to remove the inherited gene and its product itself.

Furthermore, high-density culture further inhibits transcription from the alcohol oxidase promoter. This results in the problem of increased ethanol by-products that reduce photographic efficiency.

Therefore, a relatively wide-density, non-recombinant wild-type yeast culture continuous system The results that may be obtained by the previous researcher during the When using a replacement system, it has no relation to research results. From this, the conventional technology For example, this suggests the results of high-production engineered cultures in recombinant host systems. It cannot become a foundation for

The present invention (1) Nutrient medium contains high concentration of polycarbon with little or no methanol source for a period sufficient to increase the density of viable yeast cells in the growth medium. , a high growth process in which no recombinant gene product is expressed or produced (2) the yeast host a limited amount of polycarbonate for a period sufficient to disinhibit the methanol metabolic pathway of providing source nutrients (3) Fermentation while maintaining low polycarbon carbon source nutrients and increasing methanol concentration. Because it allows the culture to maintain high production of the recombinant gene product Culture recombinant methyl hydroxide using mixotrophic feeding, cell proliferation, and gene induction methods. An unpredictable approach that allows for the continuous acquisition of increasing quantities of recombinant proteins from trophic yeast hosts. It is based on good results.

Therefore, the present invention provides recombinant gene products from recombinant methylotrophic yeast host cultures. It is based on a method of increasing production in which the gene product is by the expression of a recombinant gene sequence that is operably associated with a le-responsive expression control element. The method is (a) The nutrient medium contains a high concentration of polycarbonate with little or no methanol. Sufficient period of time to increase the density of viable yeast cells in a growth medium consisting of a carbon source. (b) A high growth process during which the recombinant gene product is not expressed or produced at all. a limited amount of multicarbon charcoal for a period sufficient to disinhibit the main methanol metabolic pathway providing source nutrients (c) Fermentation while maintaining low polycarbon carbon source nutrients and increasing methanol concentration. Because it allows the culture to maintain high production of the recombinant gene product The culture of recombinant methyl chloride using mixotrophic feeding, cell growth, and gene induction methods of culture Consists of culturing a vegetative yeast host.

Manufacture recombinant gene products by culturing microorganisms engineered to produce the product As is well understood in the industry, products made in accordance with the invention are usually fermented. The cell-free culture medium containing the recombinant gene product is recovered from the mother culture and purified. In some cases, it can be used without separating the product from the culture medium, or if the product contains a recombinant gene product. The cell or part of the cell (e.g. its membrane fraction) separates the product from the cell or part thereof. If the recombinant gene product can be used without separation, and if it contains both cells and culture medium, There are cases where it can be included in one or both of them and used directly. from yeast culture Recovery and purification of recombinant gene products can be accomplished using a number of methods well known to those skilled in the art. It can be carried out. In a recombinant methylotrophic yeast host, the method of the invention It is practiced to improve production of offspring products and its hosts are known in the industry. , a gene sequence encoding a recombinant gene product, and a gene sequence encoding a recombinant gene product. methanol-responsive expression, which is operably associated with gene sequence and expression for Transform methylotrophic yeast with the recombinant DNA consisting of the regulatory elements, and transform It is obtained by a known method, which consists of selecting yeasts that have been tested.

Figure 1 shows methanol only (white square), 4:1 glycerol-methanol mixture ( (black square), 2:1 glycerol-methanol mixture (black diamond) or limit Mut-(meth) with glycerol/non-limiting (NL) methanol mixture (white diamond) Bovine lysozyme in an induced state when 1 liter of a 1 liter fermented strain The concentration of C2 is plotted against time.

Figure 2 shows methanol only (white diamond), limited glycerol/non-limited (NL) meth Mut-strain with alcohol mixture (open squares) or under limited methanol usage. In the induced state when 1 liter of 10 Mut strains (black squares) grown in The concentration of bovine lysozyme C2 is plotted against time.

Figure 3A shows that when 1 liter of Mut- strain was fermented with only methanol (white square), The concentration of bovine lysozyme C2 in the induced state of 10 liters of fermented black square) This is a plot of degrees versus time.

Figure 3B shows 1 liter of Mut-strain with mixed glycerol-methanol feed. It is in the induced state when fermented (white square) and when 10 liters are fermented (black square) Lysozyme concentration is plotted against time.

Detailed description of the invention The present invention demonstrates that the major alcohol oxidase in a recombinant Pichia pastoris yeast host A model simulation of bovine lysozyme C2 production under the control of the (AOX) gene promoter. Draw it as a stem and describe it more specifically. autonomously replicating plasmids and episomes Alternatively, the chromosomes may contain a wide variety of desirable recombinant products (e.g., methyl nutrient has DNA encoding one of the following: polypeptides that are foreign to the yeast host , whose DNA is expressed under the control of methanol-responsive regulatory elements. It has been found that the invention is equally practiced using one of the chillotrophic yeast hosts. There is. These desirable products include human lysozyme, human peroxidoreductase, human There are epidermal growth factor (EGF) (1-52) and (1-48).

In its preferred W3 mode, the present invention, which is a mixed feeding method, adds glycerol to the breeding charcoal. Using methanol as a carbon source for both growth and induction of heterologous gene expression .

The two feedstocks work together to allow for more efficient cell growth and heterologous gene expression. make it possible.

The fermentation mixed feeding method is an advantageous method for the propagation of both Mut+ and Mut- strains; Each has a complete and incomplete AOX1 gene. A functional AOX1 gene is missing. Therefore, Mut- strains have a harder time growing in methanol than Mut+ strains. Very slow. This property requires fermentation treatment of the recombinant Mut-strain and methanol When Mut is the carbon source, it takes much longer time than Mut. Mixed feed dispatch One advantage obtained with the fermentation method is that Mut-strains are more time-effective, i.e. It has become possible to multiply and synthesize products with quantitative productivity. continuous In fermentation, the quantitative productivity of Mut+ and Mut- strains can be increased by using mixed feeding. Ru.

The thermal work and acidity required for fermentation while maintaining relatively high levels of heterologous gene expression The fermenter feeding method is advantageous for both Mut+ and Mut- strains as it reduces the It is. Because most fermenters have limited heat and oxygen transfer capacity, and Since cell density and productivity are related to the heat and oxygen transfer capacity of the fermenter, Cooling and reduced oxygen demand should all result in an increase in cellular productivity capacity.

Strains grown using the invented method were also faster with mixed feeding than with methanol alone. to proliferate.

Mixed feeding methods for cell growth and gene induction can be used in both continuous and pre-feed fermentations. It is operable.

The method for growing Mut+ and Mut- cells in mixed-feed fermentation is briefly described below.

Fermentation takes place in three major stages: first, 2% glycero Cells grown in YNB (yeast nitrogen base) containing 4% Inoculate a fermenter containing a thicker glycerol medium. In this case, AOX 1 promoter is suppressed, so no recombinant products are produced. cell energy Ghee is instead directed towards increasing cell density.

After the cell density reaches the desired level in glycerol depletion, the cell density is 1-10-container width approximately 35g, /1 (dry wt), 20g/I in a 2L container. , the cells are initiated into a glycerol-limited mode of fermentation. This glycerol The supply lasts about 4 to 12 hours, usually about 4 to 6 hours, and the AOX1 promo allows the motor to gradually move from inhibition to a fully disinhibited state.

The mixed feeding process of the pre-feed fermentation usually starts at 30 hours, preferably about 40 hours. The above is continued, but the methanol supply is, for example, 4:1 or 2:1 (glycerol (MeOH) ratio. Glycerol and methane supplied Both tanols contain 12w+l/L of YTM and TM trace metals.

The preferred method for Mut-cells is a 2:1 ratio of glycerol and methanol. In contrast, the cells were fed to the fermenter for about 8 hours after transitional R, and the cells proliferated normally and AOX1 Ensure that the promoter is completely derepressed. Then, methanol supply rate was increased such that the residual methanol concentration was between 0.1 and 0.8%, while Glycerol supply is constant.

After the mixing and feeding process of pre-feeding fermentation, continuous fermentation is started by starting the basic salt feed. It is possible to Soup and whole cells are MeOH, glycerol, basic The capacity of the feeder remains constant as the added rate of salt is transferred from the feeder. Ru. (The outflow rate determined by the feeder volume is a characteristic known as the dilution rate. Give a characteristic value. ) Compared to growth in methanol-enriched form, Mut-strains consistently increased during the mixed feeding process. It is possible to achieve higher productivity. In Mut ten stocks, the mixed feeding process is , with productivity comparable to, or even higher than, other propagation methods.

Implementation date , Sym1-C strain Encodes bovine lysothiome C2 (the most abundant lysozyme in the bovine abomasum) cDNA was injected into gtlO using polyA+ RNA isolated from bovine abomasal tissue. It was isolated from a prepared cDNA library. Bovine Rhizothium C2 gene A partial genomic clone consisting of exon 2.3 and part of exon 4 was cloned with pLl. Screened Braley. Positive clones are complementary to the 5′ end of exon 2. Confirmed by oligonucleotide screening. One of the clones, BL3 As a result of determining the nucleotide sequence of the insert, the nucleotide sequence was found to be the full length of bovine rhizothium. An initial sequence that encodes a nucleotide sequence but shows only a partial signal nucleotide sequence. It was found that ether methionine was missing. The missing signal base sequence Add a sequence to the insert using site-directed mutagenesis to identify the missing nucleotide. Sequences were obtained by direct sequencing of abomasal mRNA to proproteins.

Mutagenesis techniques were also used to generate the complete signal sequence and mature protein of bovine lysozyme C2. EcoRI sites were placed at the 5° and 3' ends of the insert, which now encodes protein. Added. EcoRI fragment cut with EcoRI Pichia expression plasmid pAO804 inserted into. (The structure is described later.) Plasmid pA0804 is created by inserting the BglII cut vector into the AOX1 site. P, which is used for the purpose of destroying the It consists of the base sequences of The plasmid also carries the P. pastoris AOX1 transcriptional promoter. terminator and terminator-1P, Bastris HIS4 gene, reproduction and selection in bacteria The only EcoRI site, which also contains the necessary nucleotide sequences, is located between the promoter and terminator regions. By separating the region, a heterologous gene can be inserted into this region.

Plasmid pAO804 was created as follows: pBR322 was o Cut with RI and insert the protruding end into E. coli DNA polymerase I The DNA was recircularized using T4 ligase. Recirculation The transformed DNA was used to transform Escherichia coli MC1061 to ampicillin resistance. , transformation with a plasmid of approximately 4.37 kbp in size without an EcoRI site. The body was screened. Such transformants were selected, cultured, and pBR3 The nucleotide sequence of the EcoRI site of pBR322, designated as 22ΔRI: 5°-GAATTAATTC-3' 3'-CTTAATTAAG-5゜ A plasmid was produced that replaced the .

Cut pBR322ΔRI with Pvull and use T4 ligase to cut the resulting blunt ends. base sequence 5°-CAGATCTG-3° 3'-GTCTAGAC-5゜ The linkers were connected. The DNA is recircularized, also using T4 ligase. Cut the shiso with f&BglI and recircularize using T4 ligase to obtain one Because the above linker connects to Pvull-cleaved pBR322ΔRI, Many of the BglII sites created by this procedure were removed. Many Bgl Transforming E. coli MC1061 to ampicillin resistance using the treated DNA did. A transformant with a plasmid of approximately 4.38 kbp containing the BglI [site] was Screened. Such transformants can be selected, cultured, and used in future work. A plasmid designated pBR322ΔRIBGL was produced. pBR322ΔR IBGL has a base sequence of 5'--instead of the Pvuf1 site of pBR322ΔRI. CAGCAGATCTGCTG-3'3'-GTCGTCTCGACGAC- pBH322ΔRIBGL is equal to pBR322ΔRI, except with 5' .

pBR322ΔRIBGL was cut with 5alI and Bgl to create a large fragment (approximately 2° 97 kbP) was isolated. Plasmid pBSAGI5I was issued as a European patent application. It is written in line No. 0226752, and it is absolutely correct in BglII and XhoI. Cleaved P, bustris AOXI downstream region AOX1 gene terminator (AOX A fragment of approximately 850 bp was isolated from (relative to the direction of transcription from the I promoter).

BglU-Xho! from pBSAGI5I! Fragment and pBR322ΔRIBGL? The approximately 2.97 bp SalI-BglI fragment was ligated and ligated with T4 ligase. I got a game. Ligation uses the mixture to unlink E. coli MC1061. Transformed to be resistant to picillin, approximately the expected size of the BglII site (3, Transformants with a plasmid of 8 kbp) were screened. This plasmid The code was designated as pAO801. 5aI1 site from pBR322ΔRIBGL fragment The overhanging end of the 850bp pBSAGI5I fragment was ligated with the overhanging end of In the process, both the 5alI site and the XhoI site in pAO801 were was removed.

pBSAGI5I was cut with its tIiClaI and an approximately 2.0 kbp fragment was isolated. Ta.

2. The oicbp fragment is approximately 1.0 kbp of the P. pastoris AOXI promoter. , a portion consisting of the transcription start site, approximately 700 bp of hepatitis B virus surface antigen (“HB sAg”), P, bustris AOX gene polyadenylation A region of about 300 bp consisting of a signal, a site coding part, and a terminator. have a share. The HBsAg coding part of the 2.0 kbp fragment is A, OX1 promoter At the end, adjacent to a 1,0 kbp site with an EcoRI site, the AOX1 tar Adjacent to the 300 bp site containing the minator and bordered by a 5tuI site. 1.0kbp promoter-containing part, 300bp terminator-containing part? From this perspective, transcription from the AOXI promoter can be manipulated to control the expression of HBsAg. It has a small portion encoding HBsAg, oriented and positioned such that P The EcoRI site that connects the promoter portion to the HBsAg coding portion is (AO Considering the direction of transcription from the XI promoter) transcription initiation of the AOXI promoter Just upstream of the signal code triplet.

Bounded by a 2.0 kbp promoter, part of a terminator, and a ClaI site. For details about the pBSAGI5I fragment, see European Patent Application Publication N0902. 26846 and Ellis et al. (Ellis) Mo1. Ce11. Biol, 5. 1111 (1985), cut plasmid pAO801 with CIaI, Approximately 2.0 ml from pBSAGI5I by ligation using T4 ligase. The bounding fragments were ligated at a 0 kbp ClaI site. ligation mixture was used to transform E. coli MC1061 to ampicillin resistance, and the transformants were After cleaning and cutting with C1aI and BglU, 2.32kbp pBR3 There are 22 replication origins and an ampicillin resistance gene) and approximately 1.9 kbp, 1.48 The expected size is approximately 5.8 kbp), resulting in fragments of kbp and 100 bp. A plasmid was obtained. When cut with BglI[5EcoRI, the plasmid becomes 3 00bp A portion of the AOXI gene terminator and approximately 2.0bp of the HBsAg code portion. 4kbp fragment; AOX1 site (900bp EcoRI-Bgl mentioned at the beginning) AOX of the AOX1 gene (further upstream from the AOXI coding fragment than the lI fragment) an approximately 900 bP fragment containing the upstream portion of the I protein coding portion, and pBR3. Approximately 100 bp of the origin of replication derived from 22, the ampicillin resistance gene, and the AOX1 site A fragment of approximately 2.42 kbp containing the C1aI-BglI portion was generated. This plastic The Smid carries the C1aI fragment of pBSAGI5I in the desired orientation and in the opposite direction. In the wrong direction, about 3.3kbp, 2.38kbp, 900bp EcoRI -Bgl ■ There is a fragment.

One of the transformants carrying the desired plasmid, designated pAO802, was used for further processing. The plasmid was selected for this purpose and cultured to produce its plasmid. pB in pAO802 The desired orientation of the CIaI fragment from SAGI5I is the AOXI residue at the AOX1 site. The Bgl fragment at the end of the 800 bp fragment derived from the downstream of the gene is The AOX1 site of the linearized plasmid was inserted into the P, B. bustris chromosome. It has the AOXI gene in the AOX1 site that is oriented in the direction that will cause it correctly. .

pAO802 then contains the HB fragment bounded by EcoRI and 5tuI sites. I removed the sAg code part. Cut the plasmid with 5tuI and '-GGAATTCC-3' 3°-CCTTAAGG-5' The blunt ends of the linker having the base sequence were ligated using T4 ligase. The mixture is After that, it was treated with EcoRI and ligation was performed again using T4 ligase. .

Transform E. coli to ampicillin resistance using ligation warm The transformant has the expected size (5,1 kbp) of approximately 1.78 kbp, 900 b p, 2.42 kbp EcoRI-Bglll fragment, approximately toobp, 2.32 k bp, 1.48 kbp and 1.2 kbp BglII-C1aI fragments. We screened for lasmids. This plasmid is designated pA0803. Transformants with the desired plasmid are selected for further manipulation, cultured and p. A.

803 was produced.

Plasmid pA0804 is derived from pA0803, and pBR322 of pAO803 Derived from Bam Contains approximately 1 site of 5kbp P, Pastoris HIS4 gene It was created by inserting a fragment derived from the chromosome of P. bastris. Frelag et al. (Cre gg) Mo1. Cel　1. Biol, 5.3376 (1985) and Europe See Patent Application Publication Nos. 0180899 and 018867. pAO803 It is cut with BamHI and contains the HiS4 gene. Bgl The pieces were combined and the mixture was ligated using T4 ligase. Rai The gation mixture was used to transform E. coli MC1061 to ampicillin resistance. The transformants were cut with 5slI to the expected size >7.85 kbp. The resulting plasmids were screened. One of these transformants was used for further processing. The plasmid selected for this purpose was designated pAO804. pAO804 is approximately 1.5 kbp, and a 5.0 kbp 5alI-CIaI fragment, approximately 1.3 kbp. It has a C1aI-C1aI fragment of HIS4 gene in the plasmid. The transcription direction is the same as that of the ampicillin resistance gene, and the AOX1 promoter It is shown that the direction of transfer from

The direction of the HIS4 gene in pAO804 depends on the function of the plasmid or the AOX1 gene. cDNA coding part inserted into the dark EcoRI site of the promoter and termination factor region is not important for the function of its derived plasmids.

Pichiabastris strain GS], 15 (h i 54) - Frelag et al. Mo1 ecular and Ce1lular Biology 15.3376 ( 1985), ATCC No', 20864- is bovine lysozyme expression plasmid p A cleavage fragment of sL12A was used as the host strain. Digem et al. (Digem) D See ev, Industr, Microbio+, 29.59 (1988).

The final expression vector containing the bovine lysozyme gene is the psL12A vector. was used for the development of both Mut' and Mu7 transformants. “Mut” Use of tanol. In the Mut’ strain, the AOXI gene continues to function, so methanol Propagation in wild plants is similar to that of wild-type plants. Mut'' strain, growth on methanol was P.

The alcohol oxidase gene (AOX2 gene) with low bustris functions. However, the growth is much slower than that of the Mut” strain on methanol. stomach.

psr-i2AeBg111, BglI [fragment was extracted with whole cell lithium chloride form] G511.54 cells were transformed using a transformation system. (Flerag et al., Mo 1ecuiar and Ce1lular Biology, 5.3379 ( 19,85)), Mut- cells are recovered from this transformation. Typical Mu t” strain was called A37.

Mut” transformant has psL12A in one place in the plasmid in the HIS4 gene. In this whole fFM cell transformation method, cut S a I. It was developed using a plasmid cut with I. Mut'' cells have this shape Recovered from transformation. A representative Mut strain was called Ll.

The strain is Yeast Nitrogen Base (YNB), which does not contain amino acids (D ifco) Inc. (Labs) Detroit) + 2% glucose agar every month. On the contrary, it was retained. The inoculum is free of amino acids at 30°C and 200 rpm. Grown in 2% glycerol + phosphate buffer (pH 6,0) YNB.

Determination of spore-bearing nectar degree To minimize cell density, centrifuge the entire soup in the fermenter for 10 minutes at 4000g to keep it moist. Durum per liter of cells is calculated from measuring and determining the importance of cell sedimentation. Served. A correlation factor of 1/4 is used to calculate dry cell concentration.

Technique Z two-stop degree tip Ethanol concentration is determined by standard gas chromatography techniques.

1j-Ken to the Emperor's Day The lysozyme concentration was calculated using freeze-dried Micrococcus lysodecticus (Micr). ococcus tysodekticus) cells are used. 100mM phosphorus Determined by turbidometric assay in acid sodium buffer pi (5i, O) It was done. -Sugar (Shngar), Biochem Biophys Ac ta.

8.302 (1952), the linear decrease in absorbance (at 450 nm) at 30°C. The rate of decline of one fermenter sample measured over a period of 1 minute directly from the previously produced recombinant bovine Lysozyme C2 - Dobson et al., J. Biol, Chem, 25 9.11607 (1984)-, the concentration was calculated.

Cow 1cm The concentration of bovine lysozyme (mg/L) was calculated from the cell-free soup. total lysozyme (mg) Amounts were calculated based on the total soup containing cells from the fermenter. mass production The yield (mg/L-h) depends on the amount of liquid in the fermenter and the time of induction state. It was produced by dividing total lysozyme. When under inhibition and disinhibition The time period for these fermentations is approximately 24-30 hours. Lysozyme production per cell The amount (mg/g) is calculated by plotting total lysozyme against whole cells and calculating the slope of the plot. It was determined and produced using linear regression. The slope is the amount of gluezozyme produced per cell. Think of it as. The correlation coefficient of the slope is 0.95 or more.

−ゝ　Bi゛　・ 2 liter fermenter (L, H, fermentation 1. Haywavd CA; Biolaf 1tte>, LSL Biolafi Isoprine Stone.

N, J) in 225 ml of 10x basic salt (42 ml/n+I85% phosphoric acid, 1. 8811 Calcium Sulfate 2H20,28,6g/I Potassium Sulfate, 23.4g /l magnesium sulfate heptahydrate, 6.5g/l potassium hydroxide) and 30g glycerol Autoclaved in 700 IIll capacity containing rolls. After sterilization, 3ml of YT M, trace salt solution (5゜0ml 17'ml sulfuric acid, 65.0g, /I iron sulfate 7 water copper sulfate pentahydrate, 20.0 g/I zinc sulfate heptahydrate, 3 ．． 0 g/l manganese sulfate monohydrate fJL 0, 1 g/l biotin) was added. Then, add concentrated ammonia water to adjust the pH to 5°0, and then adjust the pH to 5°0. 0.1% Struiktol J673 Antifoam (Struiktol) ktol, Co, Stow, Ohio.

It was adjusted to 5.0 by adding 20% aqueous ammonia containing (USA). Excessive foaming , detected by a foam probe and with the addition of 2% Structl J673 antifoam. adjusted. The fermenter was then inoculated with an inoculum amount of 10-50+sI.

Due to initial glycerol consumption, the glycerol supply as described below is It was damp.

The dissolved oxygen of the fermentation was increased by increasing the air flow rate to 31/min and 1500 rpm during the fermentation. Air saturation was maintained above 20% by shaking at a speed of up to 5η.

101 fermentation (in a 14I Biolaffitch fermenter) was carried out using a Mut-mixed feed group. 7. Method includes 10x basal salt of 1,91 and 300g glycerol. O1 capacity? Starting from 5.0 liters of basic salt and 360g glycerol, or Mut-meth For the Nord mixed feed group method, 3.2 liters of 10x basal salt and 480 g glycerol Start with an amount of 8 lindres containing the standard amount. After sterilization, add 129ml of YTM and trace salt. Then, adjust the pH to 5.07, and continue to add ammonia gas throughout the fermentation. In addition, g= was set to 5°0. For excessive foaming, use 10% Struktoll J673 antifoaming. This was controlled by adding an agent. Fermentation vessels were inoculated with a volume of 200-500 ml. initial green Consumption of celol started feeding as outlined below. Dissolved B element is fermented ・Increased air flow rate up to 10 l/'min, shaking up to 11000 rpm, and/or maintained above 20% by pressure on the fermentation vessel up to 1°5 bar. .

Mut-NL　MuΔ゛゛haha After the glycerol group state is completed, 12ml/l YTM, 50% with trace salts The feed of glycerol (by weight) was 5.4 ml/h, 10 liters for 21 fermentation vessels. The fermentation vessel was started at 54 ml/h. After 6 hours of glycerol feeding, Reduced Serol feeding to 3.6n+I7'h (36ml/h, 10 liters) 12ml, /IYTM, methanol containing trace salts is supplied from 2 liters. I started with 1.1 m1/h for the fermentation vessel and 11 m1/h for the 10 liter fermentation vessel. . After 5 hours, the methanol feed is preferably reduced to zero until the residual methanol concentration is approximately 1%. ．． It was adjusted to be between 2 and 0.8%. Fermentation involves methanol and glycerol The feeding takes place for 40-50 hours.

Mut-2: 1’ h-”’ft” 1 vertical 2:1 fermentation requires methanol supply During fermentation, the amount of glycerol: methano did not increase as much as 1.1 ml/hour. Proceed as in the NL fermentation, except that the ratio (by weight) of the fermentation mixture is 2 = 1. It was.

4:1 lol to people Δ The 4:1 method is started in the same way as the NL fermentation. glycerol/methanol During co-feeding, glycerol feed rate used in 2:1 method in 4:1 fermentation The concentration is doubled, i.e. 4=1 glycerol to methanol by weight throughout the fermentation. 7.2ml/hr for IL and 72ml/hr for 10L so that the ratio is The fermentation is a Mut-employed 4:1 mixed feed fermentation for both IL and 10L fermentations. was done following the same method.

Mut-methanol After the glycerol group state is completed, the state before induction feeding is changed to the residual methanol concentration to 0. ．． By adding methanol to the fermenter to maintain between 2 and 0.8% I started to When carrying out 10L, YTM and trace salt are not used. Instead, 40 w+I IM+trace salt (5o+I/] sulfur acid, 4.8 g/l iron chloride dihydrate, 2 ．． 0g/l zinc sulfate monohydrate, o2g/l boric acid, 0.2g/l molybdenum sodium acid, 0.3 g/l [manganese acid monohydrate, 0.08 g/l potassium iodide] Copper sulfate pentahydrate) and 2 mg/l biotin every other day. Injected into fermenter.

Mut”menol-Δ After consuming the glycerol, add 12 mg/l + y’rM, 50% glycerol with trace salts. Feed reserol to 2 liters to 12ml/12ml or 10 liter fermenter It started at 200 m1/h and ran for a total of 7 hours. 6 hours of glycerol feeding After that, feed 2 liters of methanol containing 12 mI/l YTM4 trace salt. 1.1 ml/h for the fermenter and 11 ml/h for 10 liters in 5 minutes began. An increase in dissolved oxygen is seen after the methanol feed is shut off. When this occurred, the methanol feed was restarted for the next 5 minute interval. The latter process can be described as meta How many times until the dissolved oxygen is observed to react immediately upon discontinuation of the nord feed? Once this occurs, the methanol supply is increased to 20 methanol per hour at 30 minute intervals. % increased. For methanol feeding, the feeding rate is 7 liters per 2 liters of fermenter. ．． 6 ml/h, increasing until reaching 126 ml/h per 10 liters. So Fermentation takes place for 40-60 hours per 2 liters of fermenter, ° per 10 liters. It was carried out for 25-35 hours. Soup wo゛(Yai゛゛) 400m1 IOX basic salt, 80g glycerol and deionized water (to 1 liter) A 2 liter LH fermentor was sterilized. After sterilization and cooling, add 3ml YTM4 solution. Then, 60 ml of A Mut-cell inoculum was inoculated and the pH controller was set at 5.0. During swarm growth, shaking How to cycle the speed to maintain the dissolved oxygen partial pressure above 20% air saturation. It was adjusted. 12 mg/l YTM after initial glycerol consumption.

50% glycerol solution is pumped into the fermenter at 20 mg/h. did. After 4 hours, the glycerol feed rate was reduced to 10 mg/hr and 12 m Feed of methanol containing g/I YTM' was started at 1.0 ml/h. 3 After hours methanol alone doubled. After 90 minutes, methanol feed rate of 2 ml/h 13.5 hours after adjusting the temperature to 3.8 ml/h and starting methanol feeding for the first time. It was kept constant until after r hours.

Mut-combination delivery, culture 3.2L IOX basic salt, 800g glycerol, 32ml YTMJi amount salt YNB+2 in a 14L Biolaffitch fermentor containing 7.6 kg sterile medium. % glycerol ÷ phosphate buffer inoculated with 500 ml of overnight culture of A37 strain. Ta. Fermentation was carried out at 30°C, with pH controlled at 5 by the addition of NH, to avoid excessive foam. Standing is controlled by Sudractol J-673 antifoam.

When the initial group load of glycerol is consumed, 12 m1/l YTM, and 12 ml / II M + 50% glycerol containing trace salt feed for 200 m It started at 1/h. After 6 hours, reduce glycerol feed to 122 m1/h Then, methanol feeding was started at a rate of 19 ml/h. Start methanol feeding After 8 hours, the amount in the fermenter was reduced to 8.4 with an automatic weight controller, including 4x basic salt feeding. Controlled to L. After 4 hours, all feed rates were lowered depending on the ethanol accumulation. It has been adjusted in this direction.

Adjusted feeding speed is 60m for salt, methanol and glycerol each 1/h, 14 m1/h, and 32 m1/h.

After 20 fermentations at the above flow rate, reduce the volume to 7.4L and reduce the speed to 156L. m1/h, 41 m1. /h, increased to 88m1/h. Next day delivery 1 Decrease to 19m1/h, 30m1/h, 65m1/h and keep it constant for 2 days. there was. The final feed adjustment is to adjust the flow rate to 80ml/ml for salt, methanol, and glycerol. h, 30 m1/h, and 36 m1/h for an additional 2 days. Communicating When the feed rate was adjusted downward for the first time after entering the continuous state, the inside of the fermenter Lysozyme concentration ranged from 110 mg/l to 525 mg/l at final sampling. In a continuous process, increasing yields resulted in a quantitative productivity of 10 mg/lh.

M　u　　　　　　゛゛゛゛゛゛ When 14L fermentation was carried out with "Mut" strain, lysozyme production was increased by manipulating the feed composition. It was also found that productivity was promoted. Glycerol:methanol ratio from 4:1 to 2: 1, the quantitative productivity increased from 125 mg/lh to 9 mg/lh. . Quantitative productivity is due to lysozyme values calibrated by cell density You have to be careful about that. Productivity is rather per liter of cell-free soup Based on the amount of Norizozyme per liter of fermenter soup. I'm hot at this job The cell density in the cell-free soup is 25-40% higher than the whole soup value. It should be. Mut-, Mut” (representative culture was used.

A comparison of various issuance formats is presented in Table 1 as fermentation runs at IL scale. Ru.

Table 1 1 liter-reso-mu C1, OH supply mixed feed MUT-CH, OFI supply mixed feed HUT ~ 4: 1 2:I ML MUT-HUT"" (4:1) Maximum lysozyme concentration (mg/ I) 250 180 290 375 450 130 Induction time (hours) 17 5 39 43 45 39 49 Cell density (dry g/l) 60 82 85 103 84 78 Volumetric productivity (B/1-h) 1.2 3.4 4.8 5 ．． 6 7.7 3.0 Norizozyme per cell Yield (g/g) 5.2 2.3 3.7 4.0 5.6 2.4 Max. Nol concentration (mg/l) 10 210! 00 100 30 N, D.

The salient points found in Table 1 are listed below.

1. The volumetric productivity of the Mut strain cultured using the mixed feeding method of the present invention is The volumetric productivity is 3 to 6 times higher than that of the Mur strain cultured using the feeding method.

2. Maximum amount of bovine lysozyme secreted by Muji cells cultured by mixed feeding method The concentration is the maximum concentration (25 0n+g/l) (180mg/I), and exceeds (290 and 375 mg/I), clearly adding approximately a quarter to the concentration of these mixed delivery methods. Reached in 1 hour.

3. When Muji cells are cultured by the mixed feeding fermentation method, the same results can be obtained using the methanol-only feeding method. This makes it possible to reach higher cell densities than when culturing the same cells. moreover, This higher cell density is reached in a quarter of the time.

4. Non-limiting (NL) fermentation method and mixed feeding fermentation method were preferable for Nut- strain. .

Figure 1 shows four different methods: methanol-only feeding; Serol and methanol 4:1.2:1 (weight ratio of glycerol nimethanol) mixed feeding method, limited glycerol feeding method, unrestricted methanol ( ML) shows the production of Norizozyme in a 2 liter fermentor by feeding method. N By the L mixed feeding fermentation method, 375...g/I was obtained in 48 hours, and the original methanol The volumetric productivity was 6 times higher than that of the fermentation method using single-layer fermentation.

Figure 2 shows the methanol single feeding Hut- and mixed feeding NL Muji fermentation. The data in this figure are compared to the ``Hut'' fermentation fed with tanol alone. The productivity of proliferating Nut-cells was determined by culturing Nut-cells by the mixed feeding fermentation method of the present invention. We show that by feeding the cells, they can grow to the same level as Hut'' cells, which proliferate faster. There is. The main reason is that Nut' cells are sensitive to accumulated ethanol. Therefore, Mut- cells are easier to maintain in the fermentor than Mut' cells. , it is possible to cultivate Mut- strains in such a way as to achieve productivity comparable to that of Nut. is advantageous.

Figure 3A shows a comparison of the 10 liter and 1 liter results. higher opening Because we used starting glycerol concentrations (7% vs. 4z), the 10 liter fermentation was higher. It was expected to show product levels and productivity. With this 10 liter fermentation , reaching 325 mg/l in 125 hours (Table 2), and 2.1 mg/ih with induction stage surgery. gave volumetric productivity. A yield of 5.2 mg/g cells was obtained in a 1 liter fermentation. It was the same as the one.

The lysozyme concentration for a typical 10 liter fermentation is shown in Figure 3B. The rate of meme production slows down after 20 hours. Volumetric productivity is higher than that of methanol-supplied fermentation. is also high (5.8 mg/I-h).

Table 2 Scale-up CH30H supply MIIT up to 10 liters of 1 cow - Mixed feed (NL) MUT - Maximum lysozyme concentration (mg/l) 325 2 20 induction time (hours) 120 30 Cell density (dry g/I) 75 80 Volumetric productivity (ng/Ih) 2.1 5.8 Lysozyme yield per cell (Tsuru g /g) 5.2 2.8 Maximum ethanol concentration (mg/l) 10 220 methano Volumetric productivity increased dramatically in mixed feed (NL) fermentation compared to NL feed fermentation. ．． 2. Mioka 迭Y±：mu A plasmid called p}ILZ103 has the following sequence as Eco of p^0804. Constructed by inserting into the R1 site: 5゛-^8TTCAT(;^^G(;CTCTCATTCTTCT[;CGGC TchoGTCCTCCTTTCTCTTACI;GTCCACG(;C88CGT CTTTC^^^GGTGT(;A(:TTC:(:CCAG^8CTCTC8 ^^^(;ATT(:(:CAATG(;ATC(,CTACAGGGGAAT CA(:CCTAGC^^ACT(tl:ATGTGTTTC(:CVA8^T f;G(;AGAGT11f;TTAC^8CACACGAGCT8C^^^C TAC^^TGCTCf;AC:ACAG^8GCACT(:ATTAT(;C GAT8TTTCA(:ATC^8TAC:CCGCTACTf;GT(:T^ ^TGATII;(;C^^88CCCCΔCGAGC8GTT^^T(;CC TGTC8TTTATCCTGC8GT(:CTTT(:CTII;C^8CA T8^CATCGCTGATGCT(;TAGCTTGT(;C8^ΔCACG II:TTGTCC(;TGATCC^C^ΔGGCATTM:AGCATG( :(;TGGCATG(:AC8^8TCCTTGTC^8^^CAG8GAT GTCCGTCAC: TATGTTC88GGTTGTGG8CTGT^^G5 The difference between the first 5 bases at the end (corresponding to the EcoRI site) and the 3' end The DNA with the above sequence except for (corresponding to the EeoR1 site) is derived from human placenta. signal conjugate of prelysozyme (Castanon et al., G. ene 66, 223-234 (1988)) and human histiocytic lymphoma Signal peptide of prelysozyme of cell line U-937 (Sandström (Su) ndstrom> and Ni Isson, Intl. J. Can cer 117, 565-577 (1976j) It has a signal peptide that is the same amino acid sequence as human milk lysozyme (jojo). Jolles et al., Mol. and Cell. Biochem.　 63, 165 (1984)), lysozyme from human placenta (Castanon et al., supra) ), and the human histiocytic lymphoma cell line U-937 (Sandstron and Elci It has a mature lysozyme peptide with the same amino acid sequence as lysozyme (described above). It encodes prelysozyme.

As mentioned above in relation to bovine lysozyme c2, brerisozyme pHLZ10 3 is -P. Bastris strain [:S115, Nut1, Muji (single copy), O and Hut"" (multicopy) strain to prepare lysozyme, Human prelysozyme is produced within these cells, and mature human lysozyme is cultured. It is secreted underground. Strains that secrete human lysozyme also secrete bovine lysozyme The human lysozyme can be cultured in a variety of ways as mentioned in connection with the is produced and recovered from the culture medium. Bovine lysozyme-producing strain of methylotrophic yeast The advantages mentioned above for the mixed-feed fermentation method also apply to human lysozyme-producing strains. This applies to many people. 3. Epithelial growth factor Zupusumi top A DNA with the following sequence was constructed: 5゜-^^TTCAT(;AC^^TTCCTTTC^^TTTTTTACTC:C M:TTTTATTCGCA(:CATCCTCCCCCATTACCT(;CT CCAGTC8^CACTAC^8CAC88 (;ATC^8^CGGCAC^ 8^TTCCC(;CTC^^CCTGTCATC(;(;TTACTCM:A TTTAG^^(;(C:GATTTCGATGTTC:CTGTTTT(;C CATTTTCC^^CAC:CAC^8^T^^CC;G(;TTATTGT TTAT^^8TACT^CTATT(;CCA11;CATT(:CTCCT ^8^G8^G8^(:(:(:(;TTTCTTTG(;AT^^88G^^ ^TTCC(;ATA(;C(;A(;TGTCCTCTC:AGTCAC[; AT(;CTTACTGTCTACAT(;ACGCC(:TCTGTATGT ATATTCAGCCTCTA(:8C8ΔGTAC(;G(;TGT^8TT CCGTTGTTG(:CT8CATCGGT(;AGC(;TTGTCA11 ;T8TCGTGATCT(:^^^T(;(;TGG(:^^CTTC(;T T^88CCT(;C The first five bases at the 5' end of this sequence correspond to the EcoRI site. of this array The G at the 3' end also corresponds to the EcoRI site. Immediately 5' of the G at the 3' end of this sequence The flanking 5'-^GCTC is an artifact of the method used in constructing DN^.

Translation starting from 5゛-^TG near the 5゛ end and 5'-T^^ near the 3゛ end The remaining 417 bases ending with the triplet encoding the termination signal were Mrs. Cerevisiae (Saccharomyces eerevisiae) N-terminal 85 amino acids of Brebro α mating factor (U.S. Patent No. 4,546,082 ) and 537 amino acids of mature human epidermal growth factor of 53 amino acids! ! ! (Sunawa Then, code EGF (1-53>). Transfer this DNA to plasmid pAO804 as described for bovine and human lysozyme. The resulting plasmid, named p^0817°, was designated as P. Bastris strain (1; S11 ．． 5 and Muji, Nut” (single copy), and Hut” (multicopy) ) strains and from these strains matured by culturing with various methods mentioned above. 52 amino acid human epidermal growth factor (i.e., EGF(1-52)) and Mature 48 amino acid human epidermal growth factor (i.e. EGF(1-48>) is present in the medium. secreted. Secretion is caused by the N of polypeptide encoded by DN8 having the above sequence. 85 amino acids at the end! 2 (derived from the Brebro σ mating factor of S. cerevisiae) be mediated. The carboxy-terminal amino acids of this polypeptide are P. Bastoris It is rapidly proteolytically cleaved in the strain or in the culture medium to produce mature E(;F (1-53> is not a major component of recovered EGF. However, as mentioned above, Next, mature EG;F(1-52) was filled from the medium and slowly tagged in the medium. It is converted to stable mature EC:F(1-48) by proteolysis. (EGF (1-52) and ECF (1-48) have biological activities equivalent to EGF (1-53). Like EGF(1-53), it is therapeutically useful. ) in methyltrophic yeast The advantages of using a mixed feeding method as well as in situ production of human and human lysozyme are: Diverse Mut expression systems that produce EGF(1-52) and EGF(1-48) Similar observations were made in all cases.

E(;F(1-52) and E(;F(1-48>) are Cregg et al., Mol. Cell. Biol. 5. 3376 (1 985)) was transformed with plasmid p^0817 cut with B8+II. (The 80XI gene in the genome was destroyed by the insertion of DN8, which encodes EGF. (To obtain a Nut- strain that has been modified) P. pastoris csiis strain or The original form of p^0817 was transformed by the loblast method (P. pastoris Mut'' strain by adding P. to the homologous region in the genome). It is also produced by culturing S115 strain. Blasmid p. There are two identical expression force sets connected head-to-tail in ΔO817. exist. Each cassette is P. Pastoris eight OXI promoter, it A <430 bp Brebro a junction that is operatively linked for transfer. The above DNA encoding factor (1-.85), mature EGF (1-53), and and the downstream P. Polyadenylation signal of T. pastoris 8OXI gene Contains a file and a transcription terminator.

A prebro a junction with the above sequence of about 430 bp and an EcoR1 site at the end. A DNA fragment encoding the maturation factor, mature ECF, was isolated from 1.5 g agarose glucose. Separated. 15 μg of plasmid p^0817 (the construction of which is described below) was Digested with coR I and ligated to the approximately 430 bp fragment using a standard ligation reaction. Applicable The 430 bp fragment is in the correct orientation (Prebro α mating factor (1-85>-EGF (1- 53) Translated from the lIIRN rib'^OXI promoter encoding the M-fusion protein. To determine transformants containing plasmids that are oriented in Sumid DNA was digested with Pstl. The one with the correct structure has a fragment of about 1740 bp. occured. The correct plasmid was named p^0816.

Breproσ mating factor (1-85 >-E(;F(1-53) fusion protein expression set 1~ 8O816 was digested with BgITI and BaJI, and the approximately 1670bp fragment was excised from the gel. Retrieved from page 80816 by extracting. Next, the gel-purified fragments are It was ligated to p^0816 cut with BaIIIl'lr. The ligation reaction solution was transformed into E. coli M It was used for transformation of C1061 and aB' colonies were selected. two heads - Colonies with a plasmid containing an expression cassette attached to a P digested with stl to generate fragments of 182 bp, 1497 bp, and 9547 bp. The second plasmid identified as plasmid was named p^0817.

Blasmid p^0815 is the 80x1 inversion of blasmid p^0807 (described later). Cl downstream and near the phototerminator (see construction of p^0804 above) By mutagenizing the a1 site to change it to the BamtlI site. I built it. The oligonucleotide used for mutagenization of p^0807 was It has the following sequence: 5'-GAC(:TTCGTTTCTCCCGATCCAA TCC (:GTAGTTTAT. Mutagenized plasmid, p808 It was named 07-Bauke. Blasmid p8O804 was digested with Bi+III and 2 5ng of 2400bp fragment derived from p^0807-Ban digested with BglIT was ligated to a 5400 bp BgIII fragment of 25OnH. The correct structure is Ps After digestion with tI/BamHI, bands of 6100bp and 2100bp can be identified. It was confirmed by this. The correct structure was named p^0815.

Blasmid pAO807 was constructed as follows: containing the replication origin (ori) Approximately 458 bp RsaI-Dral DN^ fragment (referred to as “fl-ori fragment”) ) or bacteriophage f1 were obtained from the phage using conventional methods.

pBR322 (2 μg) was partially digested with 2 units of DraI. Connecting mixture It was used to transform Escherichia coli JM103 strain. A+np' transformants were collected and transferred to Duplicate dyeing with Luperphage R408 was carried out. Isolate single-stranded phages from the culture medium, It was used for reinfection of JM103. The atop' transformant is a Dra of pBR322. fl-or cloned into one site (nucleotides 3232 and 3251) Contains the plasmid pBRfl-ori, containing i.

Plus length pBRf 1-or i (1011g) with Pstl and Nde I After digestion, the approximately 0.8 kbp fragment containing fl-ori was collected on a 1.2z agarose gel. Separated by pneumophoresis. About 1100n of this DNA was digested with Pstl and Nde■ and mixed with 1100n of pAO804 which had been treated with phosphatase. This mixture was clumped and used to transform Escherichia coli JM103 to obtain pAO807.

Seedling stock 5 [Ji 20. Digest the g expression vector p^0817 with Bglll and connect it in tandem. The expression cassette was excised. Linear DNA fragments obtained by digestion (5μ g) using the spheroplast method [Flerag et al., Mo1. Ce11. Bi ol, 5.3376 (1985)] to P. pastoris strain 5115 ( ＾TCC20864) was transformed. Select the old S+ cell and change the cell's meta The growth under nol phenotype (Nut) was determined.

Approximately 151 cells were His” Mut-, and the expression vector was correctly aligned to the ox1 locus. It was suggested that the XI gene was inserted at this position and destroyed the XI gene. plasmid p Southern fraction of EcoRI digest of transformant using AO803 as probe Analysis confirmed the disruption of the HeXXI gene and indicated the number of inserted expression units. It was done. Strains selected for further studies were named as shown in Table 3.

Table 3 Soil excavation, anal preparation, and construction work 1 G-E (:F817S10 Nut-His” 8OXI IG-E to F817 S7 MujiHis” 80XI 1-E [l; F817S9 Hut-Hi s""　ΔOXI In plural expression 3, the "copy number" refers to the inserted BgII I means the number of fragments. Each BglII fragment is preproa mating factor (1-85)- Contains two expression cassettes for EGF(1-53) fusion protein.

Hut” stock P. Bastris strain G5115 (^TCC20864) by spheroplast method. Therefore, 51 Jg of uncut vector p^0817 was transformed. this In type transformation, the plasmid is transformed with P at the site of homology with the fragment on the plasmid. , inserted as an addition to the genome of P. pastoris. Transformants were His″Mu t” phenotype, and Southern analysis was performed on some of them. EcoR For I digest, plasmid pYM4 [pYM4 is pYN30 (NRRL B -15890) with C1al and religating the ends] was analyzed using the probe as a probe, and from the hybridization pattern, six Appropriate insertion was demonstrated in two of them. For further research Table 4 shows the strains selected.

Table 4 Earth excavation Artificial work Color ■ Jun Sword and I C+EGF817SI Mut"His" HIS4 1G+EGF817S6 Mut “His” HIS4 1 LL eye A0 fermenter operation start and general operations 2 liter fermenter (L, H, Fermentation Co., Hayward, C^; Olafuit, LSL Biolafit, Princeton, NJ), 225+I 10X basic salt (52 1/l 85 [1 phosphoric acid, 1.8 Fi/I calci sulfate) um-2HzO), 28.6g/I [acid! J”mu, 23.46/I sulfuric acid Gnesium-7H20), 6.5g/I potassium hydroxide) and 30g0$g It was autoclaved with a volume of 700 ml of lycerol. After sterilization, 3-sho 1 YTM4) and the pH was adjusted to 5.0 with concentrated aqueous ammonia; then throughout the fermentation, Add 2°2 ammonia water containing 0.1g Structol J673 anti-foaming agent The pH was adjusted to 5.0 by this. Excessive foam will cause the foam to reach the foam sensor in the fermenter. Conditioned during the fermentation period by adding Structol J693 in case of contact. It was knotted. Next, add 10 to 50 ml of inoculum (phosphate buffered 0.6 52 yeast was cultured with shaking overnight in a nitrogen-based, pH 6, medium containing 2$ glycerol. culture solution) was added. The point at which the initially present glycerol is consumed Then, glycerol supply was started as follows. The dissolved oxygen of fermentation is The air flow rate was increased to 3 liters/min and the shaking speed was 1500 rpa+t. By raising the temperature, the air saturation was maintained at 201 or higher.

For 10 liter fermentation (in 15 liter biorafit I fermenter), use the Hut” method. For the tanol-fed batch method, 4 liters of 10X basic salt and 520 g of glycero It started with a volume of 7.0 liters containing 100 g of water. After sterilization, YTM, and IM death Add 30 liters of Shisu salt, adjust the pH, and add ammonia gas during the fermentation period. The pH was continuously maintained at 5 by adding . Excess bubbles are 5z struct The mixture was adjusted with the addition of Roll J673 anti-foam agent. 200-500…1 culture solution in fermenter was injected. Once the initially added glycerol has been consumed, proceed as described below. We have started supplying as much as possible. Dissolved oxygen increases the air flow rate to 40 liters during the photofermentation period. /min, shaking speed to 11000 rpm and/or fermenter pressure to 1.56 bar. Air saturation was maintained at above 20% by increasing the temperature to 20%.

b. Cultivation of Muji strain in a 1 liter fermenter (1) Hut-(NL) mixed feed bar After the completion of the patch fermentation glycerol patch stage, the 12 m1/I YTM-) level was 50z (glycerol) containing 2 liter fermentor ml/h. After 6 hours of glycerol supply, the glycerol supply was increased to 3.6n. Reduced to L'l+ (36 m1/h in case of 10 liters) and 12 m1/h YTM,) Methanol supply containing race salt is 1°1 for a 2 liter fermenter. ml/h. After 5 hours, the remaining methanol concentration is reduced to about 1z. Adjust the methanol supply so that the amount is between 0.2 and O.S$. Fermentation is Periodically sample F+ and collect it 36 to 50 hours after starting methanol supply. did.

(2) Mut-methanol supply batch glycerol batch after darkest is completed , methanol was added to the fermenter to maintain the residual methanol concentration between 0.2 and 0.8z. The induction feed batch darkest was started by adding nol. From the fermenter Samples were prepared periodically and collected 167 hours after incubation with methanol.

(3) Alternative method for ECF (1-52) production 400ml110 Basic salt, 808 2 liters containing glycerol and deionized water (make up to 1 liter) I The fermenter was sterilized. After sterilization and cooling, add 1 part of YTM solution and 20 The pH was adjusted to 3.6 using XNH,OR. Add 60 ml of Mut-cell culture medium The controller was set at 5.0. Shaking speed during batch cultivation was increased periodically to maintain the dissolved oxygen partial pressure above 20% of air saturation. At first When the added glycerol is completely consumed, 12ml/l containing YTM A 5 oz glycerol solution was flowed into the fermentor at a rate of 20 I/hno.

After the 4 o'clock opening, the basic glycerol rate was reduced to 10 m1/hr and 12 nl/I Supply of methanol containing YTN- was started at 1.0 ml/h. 3 hours later, Methanol feed rate was doubled. After 90 minutes of incubation at 2 ml/h, methanol was supplied. 13.5 hours after starting methanol supply by adjusting the speed to 3.8 sl/Ii It was kept constant until later collected.

c. Growth of Muji strain in a 2 liter fermentor - Mut” methanol fed batching After the recerol has been consumed, 12 ml/l YTM=) 5 containing lace salt oz glycerol feed at a rate of 12IIIL7'h in a 2 liter fermenter. A total of 7 dark incubations were started.

6 hours after supplying glycerol, 12 +++ I/h YTM, containing trace salt. The methanol supply was started at a rate of 1.1 ml/h for 5 minutes. methanol supply If a rise in dissolved oxygen is observed after stopping the methanol supply, continue the methanol supply for an additional 5 minutes. I went between. After a rapid reaction of dissolved oxygen to methanol supply interruption is observed. Repeat the process; if this happens, turn on the methanol supply for 1 hour. Increased by 20% at 30 minute intervals. For methanol supply, the supply rate is 7.6ml. /h. Fermentation continued for 40-60 hours.

d0 fermentation results In the methanol-fed batch method, Ni-E (:F817S9 and G-EGF817S Cell growth of both strains of 10 was similar, with approximately 300 g/I moisture after 167 hours. cells were obtained. However, in multicopy strains -E (:F817S9 is 4 00+ng/I of EGF and has only 2 copies of the Ell:F expression cassette It was twice that of other stocks. The maximum concentration of EGF was grown in methanol for 120 hours. reached after setting.

In the mixed feeding method, all strains multiplied to more than 300g/1 and produced multicopy strains. The 400 mg/I of EGF produced is also lower than the level produced by 2 copies of 1 strain. it was high. These strains 1. grew almost significantly faster in the mixed feeding method.

Multicopy strains use mixed feeding compared to feeding only methanol. As a result, the methanol incubation time required for EC:F production decreased, compared to 120 hours. It was 35 hours. By initial batch growth in glycerol to build up cell mass. This adds an additional 24 hours to the total process time. Methanol supply method and mixed supply The EGF productivity by feeding method was 3 gl-'h-yuki and 7 mg l-h-yuki, respectively. 1. -1.

Mut” strain G, EGF817S1, which has 2 copies of the EGF gene, has 2 copies of the EGF gene. produced hE[;F at a concentration similar to that of the N01 strain, which has the hEGF gene.

e. Stability of secreted EGF in Pichia bastolis fermentation medium Fermentation implementation period HPLC analysis of EGF in the medium revealed that the 1-48 peptide was longer. It was shown to be much more stable than The longer form is during fermentation, after induction. Seen early. After 24 hours of culture in methanol, the 1-48 peptide accumulated and became bright. It was clearly another form of degradation product. 1-48 peptide is very stable under fermentation conditions was present and accumulated for up to 6 days even in longer fermentation methods. Don't expect this Due to its high degree of stability, this form of hE (:F) has a longer production and purification time. It's much easier than .

f, Biological activity of E(:F(1-48>) 1-48E(:F peptide, inv Both in vitro cell proliferator assays and in vivo gastric ulcer healing stimulation The activity was tested. This peptide has high biological activity in both types of tests. It was shown that there are

4. Human super sidism-ze Blasmid 5OD104 Plasmid psOD104 contains approximately 470 bp at the EcoR1 site of p^0804. It was created by inserting DNA with EcoRI sites at both ends of Yes, the DNA has been described by Hallewell et al., Nucl, ^cids. As shown in Figure 1 of Res, 13.2017-2034 (1985), 154 amino acids and 465 base pairs encoding a translation stop codon, and two examples described below. They have the same array except for the outside. The two exceptions are the psOD104 insert. The last base of the triblet encoding the stop codon is the sequence of Hallewell et al. 8, it is G, and also the triblet that codes Thr-54. Of! The base after & is G, whereas it is 8 in Hallewell et al.'s sequence. be. In psOD104, the insert in the EcoRI site of p^0804 is human Subunits of red blood cell Cu/Zn superoxide dismutase (here: , which codes for “human superoxide dismutase or “human SOD”). do.

P, bus created by transforming with psOD104 or fragments thereof. The Tris strain produces human superoxide dismutase intracellularly. these Human SOD obtained from cells has the activity of original human SOD and has no therapeutic application. SOD, including human'sOD, is useful in the same applications where it is used.

female Plasmid psOD104 contains both P. pastoris Nut'' and Nut- Used for stocks of. The host strain is G5115, which requires histidine. (ATCC No. 20864). Transformation was performed according to Frelag et al., Mo1. C. e11. Biol, 5.3376 (1985). I used the last method. To create the Hut'' strain, undigested psOD1 04 was transformed into (:5115゛) and His" cells were selected. Nine His" ” Southern hybridization was performed on the strain.The chromosomal DNA was Digested with I and containing ^OXI 5' and 3' ryoukiwo p^0803 pYM4 containing the IIS4 gene (or Pichia) was used as a probe. BglI I digestion was also performed to obtain 5°-Δ^CTCATII, which is homologous to the hSOD gene fragment;^^ Add an oligonucleotide with the sequence GCCT to CAT and C8 to G^^TCCAT. It was used as a probe. From the results of Southern analysis, the N of the three classes as shown in Table 5 ut' transformants were identified: Table 5 Soil excavation, Yu Sei Nie, late accession to the throne C-5OD104C1,4,5,7,8180XI+5OD104C2,9, 101HIS4(+5OD104C321(IN4 and 0XINut- strain created) To do this, plasmid psOD104 was digested with BgITI and 5 cells and selected His" cells.) The "Iis" strain expresses Mu. More choices were made regarding the type.

Approximately 121 transformants showed slow growth and the hex1 locus was disrupted. suggested. 22 of these were compared to Comparison of control strain G-PA0804 and G-PAO8O4 strain G, i, pAO 804 No. Bgl II digested material Tehachi is a strain obtained by destroying XI. Ru. This shows the expected Muji phenotype, but the recombinant gene product is not expressed. stomach. All hsOD Muji transformants converted to G-P almost as fast as 0804. It has been shown that they can proliferate at different temperatures. Slow growth means that heterogeneous gene products enter the cell. This suggests that it is toxic.

Southern analysis of nine His''Muji cells was performed as described above. All nine lines One copy has been inserted into the OXI locus, indicating that this gene is disrupted. It was done. These strains are named Ni-5OD104C1 to G-SOD104C9. I got kicked.

! fermentation From the results of the shake flask experiment, two Hut” strains G+5OD104C10 (1 co. P) and G+5OD104C5 (2 copies), and one Mut- strain, to- For 5OD104C5, an 11-shot fermentor for hSOD production was evaluated. Mu t” cells were grown in a methanol-limited batch method, and Mut-, IM cells were grown in methanol-limited batches. Can be grown by both overfeed punch method or mixed feed batch method: 1. Methanol Feed batch, methanol controlled, Mut” phenotypic fermenter 700 ml Basic salt medium (R final basic salt concentration is 3.3x) (10x basic salt diphosphoric acid (85$ )42.0ml/I, sulfuric acid h Lucium 4HzO1.8g/I, sulfuric acid h' J'7mu 28.6g/l, tER magnesium 7H2023,4g/I, Sterilized under high pressure with potassium hydroxide (6.587I) and 4z glycerol. Ta. After sterilization, add 3 ml each of YTM, and IM, and adjust the pH to 5 with JNIl, 0)1. tailored to. This is followed by a dilution containing 0.11 Storkutl J673 anti-foaming agent. pH 5 was maintained by adding NH,0 fl (1:4). Injection medium The nutrient solution was prepared from the selection plate and contained 21 glycerol, phosphate buffered 0 ．． 67g yeast was cultured overnight at 30°C in nitrogen base (pH 5). Culture in fermenter The cells were injected and the bi-society expansion process continued for 18 to 24 hours. substrate is depleted At that point, the 5oz glycerol supply (YTM, and IN, each at 12+al/ (including l) at 12 m1/h and continued until 7 o'clock in the morning. When the substrate is consumed , usually 6 hours after the glycerol feed, the methanol feed (YTN, and IN) is (containing 12+al/I) at 1.5nl/hr, plus 3nl of YT M, and IN were added to the fermenter. Every 30 minutes, prepare 10 g increments until the final Continue for 10 hours until the feed rate is 7.5 ml/h. The container is MeOH-induced & Collected after 48-80 hours.

2. Restricted MeOFI, continuous culture, Mut” phenotype fermentor 01' was prepared similarly to the fed batch method. Once all the glycerol is used up , 5$ MeOH supply (per liter MeOH, 4x basic salt, 12ml Y TM4 and IM) were started at 10 cocoons 1/h. 5% MeOH supply is , which increased to over 60 nl/h over the next 6 hours. The volume of the size of the plant is 1 liter (about 30 hours), keep the volume in the fermenter constant at 1 liter. The recovery stream was started at the same rate as the feed stream.

At 143 hours of incubation in MeOH, 1 ml of copper and zinc solution was added to the reaction. Ta. HeO ■ Culture 2 Culture 2 Darkness 5 to 100% MeOH supply (YTM. and IM+ 12ml/l addition). Switch from continuous culture to fed batch method by switching and terminating the collection stream. Ta. The fed batch method was run for 72 hours as described above, with a total of 50 3, methanol-fed batch, excess MeOFl, Mut-phenotype development, incubated for 3 hours. The fermenter was prepared in the same manner as the Muji methanol supply batch method described above, and the culture solution was injected. . When the substrate is completely consumed, the methanol supply (YTM. and IN, each containing 12 m + 171) at 0.5 ml/h, YTM. and IN, that Three cocoons and one cocoon of each were added to the fermenter. The feeding rate is such that the residual MeOH concentration is approximately 4 g/l. Increased during fermentation to maintain. Containers 6 to 10 days after MeOH induction It was collected in

4. Mixed feeding batch, Nut-phenotype The fermenter was prepared in the same way as the Mut methanol-fed batch method described above, and the culture solution was injected. did. Once the substrate is consumed, add 5oz glycerol (YTM. and IN, respectively) at 5.4 ml/h and continued for 6 hours. It was. Next, the glycerol supply rate was reduced to L5ml/h, and each of the three YTM ．． and r? Add L and supply one MeOFt (YTM. and IN, respectively f2ml) /I) was started. The initial rate of Neo■ addition is approximately 1 ml/h (MeOH: The cellol feed ratio was 0.7:1). Adjustment of IB increase every 30 minutes is as follows: The final supply rate is 4.9ml/h (MeOH:glycerol supply ratio 2:1) I went there for the next 10 hours. The MeOH supply rate is such that the residual methanol concentration is 4g. It was adjusted so that it did not exceed /l. Vessels were harvested 80 hours after MeOH induction.

result Mut” strain (2 copies and 1 copy) using the methanol-fed batch method. , high cell density (340-350g W/I) was reached in just 54 hours (starting time). Fermentation method 1). ('4' means 'wet weight'.) Commonly used for Hut-strains. The methanol-fed batch method (fermentation method 3) is a much longer fermentation and typically Usually lasts 6 to 10 days. For example, with this method, a cell density of 280 gMW/I It took 150 hours to reach this point. Using the mixed feeding method (fermentation method 3), Hu It takes only a short culture time for the t-strain to reach the same cell density as the Hut” strain. I was condemned. Although the cell density is 380g4/l (95g dry weight/1) It took 80 hours.

From the time course of hsOD expression in fermentation methods 1.3 and 4, the highest h Based on sOD level (3500klJ/ug, or 3.SU/pg specific activity) Then, 0.92 g/I) was produced by 2 copies of the Nut” strain. It also has one of the highest dead productivity, 240U/g1, as shown in Table 6. I111I-h was shown.

Among the 1-copy strains, Hut showed the highest expression level and death productivity. ' strain ζ'', followed by Muji strain, which was cultured on a mixed substrate. methanol limit Muji cells cultured in the fed batch method had lower productivity than the others.

Initial runs of Fermentation Method 1, as suggested by similar yields and dead productivity (Table 6). showed reproducibility in later runs of the method. The latter two of this fermentation method The run was induced with methanol for 80 hours. hsOD yield depends on induction conditions High expression levels, each 5100 kU, are proportional to the amount of cells produced under /l and 1400 kU/I were obtained with longer runs. Specific activity of hsOD Based on our method for determining sex, the highest expression levels occur in late fermentation method 1 runs. It depended on it. At this time, 1.3 g of hsO per 1 liter of fermenter volume D was produced.

As mentioned above, execution 476 is performed first in a continuous culture method and then in a fed batch method. It was conducted. In continuous culture, hsOD levels were maintained for 431 hours, and copper addition to the reaction and was insensitive to zinc addition. As shown in Table 6, this fermentation method Two copies of one strain G+5OD104C3 cultured in the same manner showed the highest mortality productivity (33 0U/g/h).

Comparing the results in Table 6 for runs 445 and 446, the benefits of mixed-fed fermentation are clear. It becomes clear.

The hsOD expression levels described above are based on data from the activity assay described below. life Because the assay measures only functional molecules, it does not contain denatured or active enzymes. The expression level shown is a lower limit; therefore, the expression level shown is a lower limit; Bell will be equal to or higher than this value.

Human SOD activity assay Nitrate assay [Y, Oyanagi, Δna1. Biochem, 142.290 (1984)] was used to measure hsOD activity. This assay is based on hsOD. It is based on the oxidation of hydroxylamine to nitric acid, which is used as an indicator dye. Detected by colorant.

The nitrate assay is very useful for determining hsOD concentrations in Pichia extracts. It has been known. Table 7 shows that some samples collected during the fermentation process show high activity. However, the control sample (G-PAO804) showed a much lower value; This assay often showed values only as low as 5z for recombinant strains; Recombinant hsOD activity could be distinguished from other SOD-like activities in the extract, as shown in Table 7. As shown, the deviation was generally less than 10%.

Fishing tanoum mommeko 441-21. 791 10.44 441-54 1.779 4.00 442-21. 385 6.94 442-54. 569 11.40 445-34 6.89 ．． 340 Number 1 is the average value of four assays.

Calculated from the standard deviation of the average value of two samples.

Nitrate assay was performed using human red blood cell SOD standard (Sigma Chemical Co.) and P, Bastris. Both cytochrome C assays and human recombinant SOD purified from and corrected for cytochrome C assay. Red blood cell SOD is available from the vendor. Therefore, the specific activity is reported to be 2.7 units/μg in the cytochrome C assay. . We determined the specific activity to be 3.8 units/IIg in this assay, and the present invention Therefore, it was found that the prepared hsOD showed equivalent values. all protein Quality concentrations were determined using quantitative amino acid analysis. Nitrate assay consists of two preparations It also showed similar values. Therefore, values obtained from unknown samples are always known By comparing the specific activity of the purified enzyme with the value obtained from the purified enzyme, it is easier and more clear. It becomes possible to use a distinguishable nitrate assay.

Purification and stability of hsOD produced in P. pastoris >902 recombinant h sOD was extracted from P. pastoris cells with glass beads in simple buffer.

Typically, 0. bIIM 50% sodium phosphate or phosphorus containing EDT^ The recovery rate after collecting pellets using potassium acid buffer (pH 7, s) was The field is 1sss or more as determined by either sex assay or western blot. It was. (Other buffers can be used if the enzyme is stable.) Recombinant hsO D is McCord et al., J. Biol, Chew, 241 ．． 6049 (1969) for red blood cell extracts. Purified from cell extract.

Hemoglobin is precipitated from red blood cell preparations by ethanol-chloroform treatment. let Similarly, many yeast proteins were removed from P. pastoris extracts. Ru. Add 25π of ethanol, which is the volume of the extract liquid, drop by drop to the stirring extract suspension. The temperature was maintained below 4°C in a salt water-ice bath. Chloroform was added similarly (first 15 z) of the extraction liquid volume to denature the contaminating proteins, and add another 1 ml of the extracted liquid volume in an ice bath. Precipitation was caused by stirring for 5 minutes, and the precipitate was removed by centrifugation.

Superoxide dismutase was further purified by a phase extraction process. 25℃ Dibasic potassium phosphate was added to the warm supernatant. 2 PO1 (heavy) for 3oz hsOD is high when dissolved in ethanol-chloroform solution (volume vs. volume). It separates from the lower layer of temperature and comes to the upper layer. Cool the upper layer to 4°C and clean by centrifugation. .

Recombinant hsOD was precipitated with cold acetone (75z of supernatant volume), centrifuged, Suspend and concentrate to a small volume. The solution was diluted with 2.5mM potassium phosphate, pI (7, Dialyzed against 8. (Instead of potassium phosphate buffer, use another low ionic strength A buffer solution near neutrality may also be used. ) This buffer is DE-52 used in the next step. or similar anion exchange resin equilibration buffer. hson is D E-52 to potassium phosphate, pH 7,8 with a gradient of 2.5IeM to 100- ? Take a picture.

When storing the extract at 3-5°C, hsOD activity is stable for at least 4 weeks. Ru. As a purified protein, recombinant hsOD from P. pastoris is 3-5 Stable for at least 8 weeks at ℃.

Deposit A culture of viable P. bustris strain G5115 was submitted to the Microbial Deposit for Patent Purposes. Under the Budapest Treaty and the provisions promulgated thereunder, the American Type Culture Collection, Rockville, Maryland, USA (“´＾ TCC") on August 15, 1987, TCC accession number 20864. is given.

The foregoing description is a more specific method that may be used in carrying out the present invention. mentioned above Although the explanation is detailed, there are some things mentioned here that limit the overall essence. rather, the boundaries of the invention are determined solely by the legal description of the claims. It will be arranged.

Engraving (no changes to the content) Mut-host mixed feeding Methanol incubation time 1 liter bovine lysozyme fermentation Methanol incubation time Methanol incubation time Scale-up of mixed feed Mut- Methanol incubation time Submission of translation of written amendment (Article 184-8 of the Patent Act) March 26, 1991 Mr. Satoshi Ue Matsu, Commissioner of the Patent Office, magnetic 1. Display of patent application PCT/US89104164 2. Name of the invention Mixed feeding recombinant yeast fermentation 3. Patent Kawahara situation Address: 92037 for California, USA. La Hora.

North Torrey Vines Road 1 '0280 Name: The Salk I Institute Biotechnology/Industrial Associates I Incoholated 4, Agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo (1) One translation of the written amendment The scope of the claims (1) Recombinant P, recombinant gene product from a culture of Pastoris yeast host a method for producing a product, the product being operably linked to a methanol-responsive promoter. It is produced by expression from DNA encoding a foreign gene. It is something that is; (a) for a sufficient period of time to increase the density of viable yeast cells in the growth medium; The yeast host cells are subjected to a high growth process, and the nutrient medium contains: Things: A non-limiting concentration of inhibitory carbon sufficient to repress the methanol-responsive promoter source, thereby suppressing the expression of the recombinant gene product from the foreign gene. those who control, and be substantially free of methanol; after that, (b) being restricted for a sufficient period of time to deinhibit the methanol metabolic pathway of the yeast host; (C) supplying an amount of an inhibitory carbon source to the yeast host cells; The cell becomes a high production process, and methanol supply is started, thereby The medium becomes a mixture of methanol and inhibitory carbon source nutrients; The ethanol concentration is sufficient to induce the expression of the recombinant gene product derived from the foreign gene. being a minute; A method comprising culturing said yeast host by the process of.

(2) The method according to claim 1, wherein the yeast host is mut-.

(3) The method according to claim 1, wherein the yeast host is "mut".

(4) The method according to claim 1, wherein the promoter is AOXI .

(5) Request that the inhibitory carbon source nutrient is glycerol or glucose. The method described in item 1 of the scope of the request.

(6) The recombinant gene product is superoxide dismutase protein. The method according to claim 1, wherein

(7) A claim in which the recombinant gene product is an epidermal growth factor protein. The method described in item 1.

(8) Claim 1, wherein the recombinant gene product is lysozyme Method described.

(9) step (C) is carried out using a glycerol:methanol ratio of about 2:1; The method according to claim 1, wherein the method comprises:

(10) Step (C) is carried out using a glycerol:methanol ratio of about 4:1. The method according to claim 1, wherein the method comprises:

(11) The process (C) is performed so that the methanol concentration does not exceed about 1% in the medium. Claim No. 1, which is carried out by adjusting the methanol feed rate. The method described in Section 1.

(12) Claim 1, wherein the culturing is carried out at about 27°C to 35°C. The method described in section.

(13) Claim No. 1 in which the promoter is the AOX1 promoter The method described in Section 5.

(14) The promoter is the AOX1 promoter and the yeast host is mut - The method according to claim 5, wherein the strain is a strain.

(15) The promoter is the AOX1 promoter and the yeast host is mut '' strain, the method according to claim 5.

Procedural amendment (formality) 1.Display of the incident PCT/US89104164 1999 Patent Application No. 510384 2. Name of the invention Mixed feeding recombinant yeast fermentation 3. Person who makes corrections Relationship to the incident: Patent applicant address Name: The Salk, Institute Biotechnology/Indust Real Associate Φ Incorporated 4. Agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 6. Subject of correction (1) Domestic document international search report stating the name of the applicant's representative -ahka1mu'PCT/US89104164

Claims (14)

[Claims] (1) Production of recombinant gene products from cultures of recombinant methylotrophic yeast hosts , wherein the product is controlled by a methanol-responsive expression regulator. produced by expression from recombinant gene sequences linked so that wherein the method cultivates the methylotrophic yeast host using a mixotrophic feed; (a) the nutrient medium is highly concentrated; high carbon content, contains carbon source nutrients but little or no methanol a culture medium in which the recombinant gene product is not produced in substantial amounts by expression. for a period sufficient to increase the density of viable yeast cells in the nutrient growth medium without (b) to deinhibit the methanol metabolic pathway of the yeast host; (c) providing a limited amount of carbon-rich, carbon-source nutrients for a sufficient period of time; Increasing methanol concentration while maintaining the nutrient solution as a low-temperature, high-carbon, carbon-source nutrient , maintaining the recombinant gene product in a high expression stage. How to be. (2) the step (c) is carried out at a glycerol:methanol ratio of about 2:1; The method according to claim 1. (3) step (c) is carried out at a glycerol:methanol ratio of about 4:1; The method according to claim 1. (4) The process (c) is carried out so that the residual methanol concentration is up to about 1%. Claim 1, which is carried out by adjusting the methanol concentration during the period. Method described. (5) Claim 1, wherein the culturing is carried out at about 27°C to 35°C. 4. The method according to any one of Item 4. (6) The methylotrophic yeast host is Pichia pastoris. oris) strain, in which the methanol-responsive expression regulator is the AOX1 promoter. The method according to any one of claims 1 to 5, wherein the method is: (7) Claims 1 to 6, wherein the methylotrophic yeast host is a Mut- strain. The method described in any of the paragraphs. (8) Claims 1 to 6, wherein the methylotrophic yeast host is a Mut+ strain. The method described in any of the paragraphs. (9) Claim 7, wherein the recombinant expression product is animal lysozyme c. and the method according to any of paragraph 8. (10) Claim No. 1, wherein the recombinant expression product is bovine lysozyme c2. The method described in Section 9. (11) Claim 9, wherein the recombinant expression product is human lysozyme. Method described. (12) The recombinant expression products are human EGF (1-52) and human EGF (1-4 8) selected from the group consisting of: The method described in any of the following. (13) Claims in which the recombinant expression product is human EGF (1-48) The method described in item 12. (14) The recombinant expression product is human superoxide dismutase. , the method according to any one of claims 7 and 8.