Agency of Industrial Science and Technology

Mitsui Chemicals, Inc.

Mitsui Toatsu Chemicals, Incorporated

Ministry of International Trade & Industry

Extra Ministerial Bureau of Ministry of International Trade and Industry

Schering Aktiengesellschaft

Masaru Honjo

Yoshio Furutani

Hiroshi Uchida

Hiroaki Shimada

Akira Nakayama

Daisuke Mochizuki

Kazuya Matsumoto

Noboru Tomioka

Naokazu Naitoh

Naokazu Naito

Kazuaki Manabe

Izumi Mita

Akiko Akaoka

Koichi Kawamura

Kazutoshi Horikomi

Takayoshi Masuda

Tsutomu Takase

Setsuo Yoshino

Yoshimoto Watanabe

Takeshi Kamioka

Keiko Fukazawa

Kazunori Ando

Michiko Hori

Miwa Ikeda

Noriaki Asada

Kouichi Kawamura

A DNA encoding 20K hGH is connected directly to a gene encoding OppA protein secretion signal, or a modified form thereof, and a DNA encoding signal peptidase 1 to construct a recombinant plasmid, is transformed by the plasmid and cells of the resulting transformant strain are cultured for secretory production of the 20K hGH in the periplasm. This method enables efficient secretory production of 20K hGH and easy isolation and purification of 20K hGH from the periplasm fraction because the level of impure proteins in the periplasm is low.

Method for secretory production of human growth hormone

Administration of an administrative amount of an authentic human growth hormone having a molecular weight of 20,000 daltons to human adults can improve body composition, stimulate lipolysis, increase serum insulin-like growth factor I or be used in replacement therapy for human growth hormone-deficient patients.

Therapeutic use of 20-kilodalton human growth hormone

A DNA encoding 20K hGH is connected directly to a gene encoding Escherichia coli OppA protein secretion signal, or a modified form thereof, and a DNA encoding signal peptidase 1 to construct a recombinant plasmid, E. coli is transformed by said plasmid and cells of the resulting E. coli transformant strain are cultured for secretory production of the 20K hGH in the E. coli periplasm. This method enables efficient secretory production of 20K hGH and easy isolation and purification of 20K hGH from the periplasm fraction because the level of impure proteins in the E. coli periplasm is low.

Efficient secretory production of a recombinant protein in the periplasm can be attained by artificially regulating co-expression of a gene coding for the target recombinant protein and a gene coding for Sec protein involved in protein transport in E. coli cells under conditions in which excessive expression of the Sec protein is suppressed.

Method for secretory production of protein

In producing a recombinant protein in the periplasm of Escherichia coli, the productivity of the target ecombinant protein can be promoted by coexpressing glutathione reductase artificially with the target recombinant protein.

A neutral protease gene of Bacillus amyloliquefaciens is cloned, which gene comprises the promoter region, the ribosome binding region, the region involved in the secretion of the neutral protease, the region consisting of the structural gene for the neutral protease, and the terminator region. Each of the regions is useful as a material for construction of a recombinant DNA used for the production of proteins by culturing a host bacterium transformed with the recombinant DNA. For example, the extracellular production of neutral protease in a large amount can be accomplished by culturing B. subtilis transformed with a recombinant DNA comprising pUB110 and the neutral protease gene.

DNA base sequence containing regions involved in the production and

A human growth hormone having a molecular weight of 20,000 daltons can be effectively secreted and produced in the periplasm of Escherichia coli by constructing a human growth hormone secretion plasmid comprising a vector DNA replicable in E. coli and a DNA fragment including a promoter, a ribosome binding site, a secretion signal coding region, which are all those of the neutral protease gene of B. amyloliquefaciens and a gene encoding 20 kD hGH placed just downstream from the secretion signal coding region; introducing the said plasmid into E. coli; and culturing the resultant transformed cells.

Process for producing 20 kD human growth hormone

Modified HV1-type hirudinin which valine at the N-terminal end of the HV1-type hirudin and aspartic acid at the 5th residue of the N-terminal end were replaced with alanine and glutamic acid, respectively. A secretion plasmid into which a DNA sequence coding for a precursor with an addition of a secretion signal of neutral protease of Bacillus amyloliquefaciens at the N-terminal end of this modified HV1-type hirudin is integrated is introduced into bacteria of the genus Bacillus and the precursor is expressed intracellularly. The modified HV1-type hirudin can be efficiently secreted extracellularly while maintaining its high thrombin inhibiting activity.

Polypeptide having thrombin inhibiting activity

Residual extracellular protease activities of a Bacillus subtilis strain can be further reduced by introducing a gene for the stimulation of extracellular protease levels into the genomic DNA of the strain. The resultant strain whose extracellular protease activities are further reduced is transformed with a recombinant plasmid for secretion of a desired protein and the desired protein can be efficiently produced and accumulated in the culture medium in a large amount. The accumulated desired protein can be easily recovered from the culture medium. For example, the accumulation level of human growth hormone secreted by a B. subtilis MT-430 strain carrying recombinant DNA phGH427 for the expression and secretion of human growth hormone can be increased five to tenfold as a result of the above treatment for reduction of the residual extracellular protease activities of the extracellular protease-activity-deficient strain.

Bacillus subtilis strain whose extracellular protease activities are

A expression-secretion vector is constructed by using a DNA sequence comprising a DNA fragment obtained by subjecting a DNA sequence consisting of a region involved in the expression of a gene coding for an extracellular enzyme of a bacterium of the genus Bacillus and a region involved in the secretion of the enzyme thus expressed to a deletion in the region involved in the secretion which can enhance the extracellular production of a protein dependent on the DNA fragment. By transforming a host bacterium with a recombinant DNA molecule formed by inserting a DNA fragment comprising a gene coding for a desired protein into the expression-secretion vector and then culturing the transformed host, the extracellular production of the desired protein in a large amount can be accomplished.

DNA sequence involved in gene expression and protein secretion,

This invention relates to a DNA base sequence, capable of increasing the amount of protein secreted by microorganisms, and its derivative sequences; a recombinant plasmid including the whole or a part of said DNA base sequence; a method for preparing the recombinant plasmid in which, when microorganisms having introduced thereinto a recombinant DNA including the desired DNA base sequence are to be separated in the process of cloning, suitable transformants can be efficiently selected by taking the amount of protein secreted out of the cells and particularly the activity of an enzyme protein as an index; and a method of microbial breeding which comprises introducing the recombinant plasmid into a microorganism to increase the amount of protein secreted by the microorganism.

DNA base sequence, method for preparing a recombinant plasmid including

A recombinant DNA molecule, a portion of which includes a vector whose replication is initiated independently of the temperature-sensitive mutant gene of a temperature-sensitive mutant strain of the genus Bacillus as host is introduced into the host. The host is then cultured at a temperature which does not completely inhibit chromosomal DNA replication. By this process, the recombinant DNA molecule can be stabilized in the host and its expression can be enhanced.

Method for stabilizing recombinant DNA molecules and enhancing the

This invention provides a method for purifying crude fatty acid esters of saccharide containing at least fatty acid glycerides as impurity, and which method comprises decomposing the fatty acid glycerides by the treatment thereof with a lipid splitting enzyme or with a combination of a lipid splitting enzyme and a reducing agent in the presence of water.

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Chiba, Japan

Masaru Honjo