Mitsui Toatsu Chemicals, Incorporated

Mitsui Chemicals, Inc.

Mitsu Chemicals, Inc.

Setsuo Yoshino

Nobuhiro Fukuhara

Yoshiyuki Nakajima

Satori Sone

Nobuyoshi Makiguchi

Maki Suzuki

Kaoru Yamamoto

Masaru Honjo

Toshihiro Oikawa

Akira Nakayama

Hitoki Miyake

Masami Osabe

Mitsuo Koito

Naokazu Naito

Kenichi Ishiwata

Tomoyuki Se

Satoru Iwamori

Amino acid sequence of L-phenylalanine ammonia-lyase

A purified N-t-butoxycarbonylphenylalanine ester preparation having an enhanced optical activity can be obtained by bringing an N-t-butoxycarbonylphenylalanine ester preparation containing an optically active compound into contact with an aliphatic hydrocarbon, extracting the optically active compound with the aliphatic hydrocarbon, and recovering the optically active compound from the resulting extract.

Process for preparing N-t-butoxycarbonylphenylalanine ester

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

Escherichia coil carrying a hybrid plasmid having been constructed by inserting a desired foreign gene into an expression vector so as to permitting expression of said desired foreign gene therein was cultured at a temperature 40.degree. C. or over so that the expression of said desired foreign gene was suppressed. This E. coil (i.e., transformant) was cultured at 40.degree. C. or over in a first process to suppress the expression of the foreign gene and to support sufficient cell growth and thereafter below 40.degree. C. in a second process to release the suppression of the expression so as to permit effective production of the foreign gene product, which resulted in high concentration of the foreign gene product in the final culture.

Method of regulating expression of a foreign gene by controlling culture

Disclosed is a cloned tyrosine phenol-lyase gene which encodes an amino acid sequence represented by the formula [I], which can be expressed in the absence of tyrosine in culturing medium. A recombinant vector containing the tyrosine phenol-lyase gene as well as E. coli transformants capable of producing tyrosine phenol-lyase are also disclosed.

Cloned tyrosine phenol-lyase gene, recombinant plasmid containing the

Escherichia coli carrying a hybrid plasmid having been constructed by inserting a desired foreign gene into an expression vector so as to permitting expression of said desired foreign gene therein was cultured in a medium containing a sugar component utilizable by the E. coli as a carbon source at a concentration of 0.3% or more so that the expression of said desired foreign gene was suppressed. This E. coli (i.e., transformant) was cultured in the medium in which the sugar concentration was maintained at 0.3% or more in a first process so as to supress the suppression of the foreign gene and to support sufficient cell growth and thereafter at less than 0.3% in a second process to release the suppression of the expression so as to permit effective production of the foreign gene product, which resulted in high concentration of the foreign gene product in the final culture.

Method of regulating expression of a foreign gene by controlling the

Escherichia coli carrying a hybrid plasmid having been constructed by inserting a desired foreign gene into an expression vector so as to permitting expression of said desired foreign gene therein was cultured at a temperature 40.degree. C. or over so that the expression of said desired foreign gene was suppressed. This E. coli (i.e., transformant) was cultured at 40.degree. C. or over in a first process to suppress the expression of the foreign gene and to support sufficient cell growth and thereafter below 40.degree. C. in a second process to release the suppression of the expression so as to permit effective production of the foreign gene product, which resulted in high concentration of the foreign gene product in the final culture.

A recombinant plasmid for the expression of phenylalanine ammonia-lyase (PAL) is constructed by incorporating therein a combined promoter comprising (a) the fusion promoter (the tac promoter) composed of the trp promoter minus 35 region and the lac UV-5 promoter minus 10 region and (b) the P.sub.L promoter of the lambda phage, the tac promoter and the P.sub.L promoter being connected so as to have the same directional property. This recombinant plasmid permits more efficient expression of in Escherichia coli.

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Yokohama, Japan

Setsuo Yoshino