Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1999-12-22
2001-10-30
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S069100, C530S363000
Reexamination Certificate
active
06309864
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an improvement in the method for production of heterologous protein comprising culturing a host transformed by gene manipulation.
1. Background Art
While a broad range of proteins useful as pharmaceuticals, such as human serum albumin (hereinafter to be referred to as HSA), which is a major protein component of plasma, and the like are produced by fractionation of body fluid, this method is confronted with difficulty in securing the starting material, and the produced preparations have a strong possibility of contamination with virus and the like. The advent of the recombinant DNA technology in recent years has enabled production of such proteins by microorganisms and cells, which encourages study and development of large scale production of heterologous protein by genetic engineering. However, the yield is still low and a large scale production has not been attainable.
The method for increasing the production of heterologous protein includes a method comprising adding a fatty acid or a salt thereof to a medium to increase production of recombinant HSA (hereinafter to be referred to as rHSA) (JP-A-4-293495), a method comprising adding a high concentration surfactant having a polyalkylene glycol group (Japanese Patent Application under PCT laid-open under kohyo No. 3-500969) and the like.
In view of such technical background, the present invention aims at increasing the production amount of heterologous protein by particularly improving culture conditions.
2. Disclosure of the Invention
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems, and found that the production amount of a heterologous protein can be increased by culturing a heterologous protein-producing host prepared by gene manipulation in a medium containing a fatty acid or a salt thereof, and a surfactant, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A production method of a heterologous protein, comprising culturing a heterologous protein-producing host prepared by gene manipulation in a medium containing a fatty acid or a salt thereof, and a surfactant, and harvesting the heterologous protein from the culture.
(2) The production method of (1) above, wherein the fatty acid has 10 to 26 carbon atoms.
(3) The production method of (1) above, wherein the medium contains a fatty acid or a salt thereof at a concentration of 0.01-10W/V %.
(4) The production method of (1) above, wherein the surfactant is a non-ionic surfactant having a molecular weight of 100-100,000.
(5) The production method of (1) above, wherein the medium contains a surfactant at a concentration of not more than 0.5 g/L.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, by the heterologous protein is meant a foreign protein which is not inherently produced by a host cell but which has come to be produced by transformation.
The heterologous protein-producing hosts disclosed in known publications and one that will be developed from now, which are prepared by gene manipulation and used in the present invention, are subject to no particular limitation as long as they can be prepared by gene manipulation and are capable of producing a heterologous protein. Specific examples thereof include a cell (e.g.,
Eschenchia coli,
yeasts,
Bacillus subtilis
and the like), an animal cell and the like, which are genetically manipulated to produce a heterologous protein. Particularly, in the present invention, the host is preferably a yeast, which may be the genus Saccharomyces or the genus Pichia. Auxotrophic strain and antibiotic sensitive strain of these hosts can be also used. Preferably,
Saccharomyces cerevisiae
AH22 strain which is a G418 sensitive strain (a, his 4, leu 2, can 1),
Pichia pastoris
GTS115 strain (his 4, NRRL deposit No. Y-15851) and the like can be used.
The heterologous protein produced by a heterologous protein-producing host is not particularly limited and preferably exemplified by HSA and the like.
These heterologous protein-producing hosts can be prepared by a known method or a method analogous thereto.
For example, an HSA-producing host (or an HSA-producing strain) can be prepared by a method using a known HSA gene (JP-A-58-56684, JP-A-58-90515, JP-A-58-150517), a method using a novel HSA gene (JP-A-62-29985, JP-A-1-98486), a method using a synthetic signal sequence (JP-A-1-240191), a method using a serum albumin signal sequence (JP-A-2-167095), a method comprising integration of a recombinant plasmid on a chromosome (JP-A-3-72889), a method comprising fusion of hosts (JP-A-3-53877), a method comprising mutation in a medium containing methanol, a method using a mutant AOX2 promoter (JP-A-6-90768, JP-A-4-299984), expression of HSA by
Bacillus subtilis
(JP-A-62-25133), expression of HSA by yeast (JP-A-60-41487, JP-A-63-39576, JP-A-63-74493), expression of HSA by
Pichia
yeast (JP-A-2-104290) and the like.
Of these, the method causing mutation in a medium containing methanol is performed as follows. A plasmid having a transcription unit to express HSA under the control of AOX1 promoter is introduced into the AOX1 gene region of a suitable host, preferably
Pichia
yeast, specifically
Pichia pastoris
GTS115 strain by a conventional method to give a transformant (see JP-A-2-104290). This transformant has a weak proliferation capability in a medium containing methanol. Thus, according to the method disclosed in JP-A-4-299984, this transformant is cultured in a medium containing methanol to cause mutation and only the strain capable of growth is recovered. In this case, the concentration of methanol is approximately 0.0001%-5%. The medium may be synthetic or natural. The culture conditions are 15° C.-40° C., about 1 hour-1000 hours.
As long as the medium to be used for culturing a transformed host contains a fatty acid or a salt thereof, and a surfactant, it is subject to no particular limitation with regard to other components, and a medium known in this field is usually used. Culturing of a transformed host in a medium containing a fatty acid or a salt thereof, and a surfactant enables increase in the production amount of a heterologous protein. In addition, the enzyme that the host itself secretes is expected to suppress decomposition of the heterologous protein.
Examples of fatty acid preferably include those having 10 to 26 carbon atoms.
Examples of the aforementioned fatty acid include saturated and unsaturated fatty acids such as myristic acid, palmitic acid, palmitoleic acid, oleic acid, t-vaccenic acid, linoleic acid, linolenic acid, linoleic acid, arachidonic acid and the like. The salts of these fatty acids are, for example, alkali metal salt such as sodium salt, potassium salt, calcium salt and the like, alkaline earth metal salts and organic amine salts, with preference given to a medium containing oleic acid or a salt thereof.
The content of fatty acid in the medium is about 0.01-10 W/V %, preferably about 0.2-5 W/V %.
The surfactant to be used in the present invention is a non-ionic surfactant preferably having a high molecular weight of from 100 to 100,000.
Examples of the aforementioned non-ionic surfactant include polyalkylene glycol (e.g., polypropylene glycol having an average molecular weight of 1000-10,000, preferably 2,000-6,000), polyoxy-alkylene copolymer (e.g., polyoxyethylene-polyoxypropylene copolymer having an average molecular weight of 100-100,000, preferably 1,000-30,000), hydrogenated castor oil polyoxyalkylene derivative [e.g., hydrogenated castor oil polyoxyethylene(20)-ether, hydrogenated castor oil polyoxyethylene-(40)-ether, hydrogenated castor oil polyoxyethylene-(100)-ether and the like], castor oil polyoxyalkylene derivative[e.g., castor oil polyoxyethylene(20)-ether, castor oil polyoxyethylene-(40)-ether, castor oil polyoxyethylene-(100)-ether and the like], polyoxyethylenesorbitan fatty acid ester (e.g., polyoxyethylenesorbitan monooleate, polyoxyethylenesorbitan monostearate, polyoxye
Kobayashi Kaoru
Kuwae Shinobu
Ohda Toyoo
Ohya Tomoshi
Ohyama Masao
Carlson Karen Cochrane
Sughrue Mion Zinn Macpeak & Seas, PLLC
Yoshitomi Pharmaceutical Industries Ltd.
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