Agency of Industrial Science and Technology

Ministry of International Trade & Industry

Extra Ministerial Bureau of Ministry of International Trade and Industry

Hiroaki Shimada

Masaru Honjo

Yoshio Furutani

Noboru Tomioka

Kazuaki Manabe

Izumi Mita

Akira Nakayama

Akiko Akaoka

Koichi Kawamura

Kouichi Kawamura

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

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.

Growing community of inventors

Mobara, Japan

Hiroaki Shimada