Agency of Industrial Science and Technology

Ministry of International Trade & Industry

Mitsui Toatsu Chemicals, Incorporated

Extra Ministerial Bureau of Ministry of International Trade and Industry

Noboru Tomioka

Masaru Honjo

Yoshio Furutani

Hiroaki Shimada

Kazuaki Manabe

Izumi Mita

Tomoko Ishii

Eriko Mine

Fumio Omae

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

By screening an animal cell transformant, which has been obtained by introducing into wild-type animal cells a wild-type DHFR gene and a structural gene which codes a desired useful substance, with a medium containing methotrexate (MTX) at a concentration of 50 .mu.M or lower, both genes can be amplified effectively within an MTX resistant strain and a stable amplified transformant can also be obtained. The screening may be repeated using an increasing concentration of MTX. It is also possible to obtain stably the desired foreign gene product in a high yield by using the thus-obtained amplified transformant.

Animal cell transformant, method for obtaining the transformant and

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

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Mobara, Japan

Noboru Tomioka