The Green Cross Corporation

Haruhide Kawabe

Hirofumi Arimura

Ken Okabayashi

Tadakazu Suyama

Yukimitsu Nakagawa

Yutaka Ishida

Kaoru Kobayashi

Masayuki Nishida

Kiyoshi Tsutsui

Masami Miura

Hiromichi Mukai

Hajime Horii

Kazuo Ikegaya

Kazumoto Hirabayashi

Hideyuki Oi

Sadao Ueda

Naofumi Hayasuke

Hitoshi Minamino

Kohji Murakami

Muneo Tsujikawa

Kazumasa Yokoyama

Takao Ohmura

Koji Murakami

Toshizumi Tanabe

Shusei Uno

Ryuji Hiramatsu

Yasuo Amatsuji

Masanori Nagai

Masaaki Hirose

Atusi Masaki

Atsushi Masaki

Miho Shimizu

Hazime Horii

A plasmid for a yeast host, the plasmid comprising in sequence: (1) a yeast derived promoter, (2) an albumin-encoding region placed under control of the yeast-derived promoter, (3) a transcription terminator and (4) a sequence homologous to a part of the yeast host chromosomal sequence such that the plasmid is capable of being integrated into the yeast host cell chromosome and wherein the plasmid is incapable of autonomous replication in yeast cells; a yeast host transformed with the above plasmid; a method of producing a yeast transformant which comprises transforming a yeast host by integrating at least two different plasmids each having a sequence homologous to a part of the host yeast chromosomal sequence one at a time into the chromosome of the yeast host cell; and a method of producing albumin which comprises cultivating the above transformant and recovering the thus-produced albumin.

Albumin gene-containing plasmid, transformant carrying same, production

A methylotrophic and glucotrophic mutant strain capable of producing a heterologous protein and a method for producing a heterologous protein, comprising culture of the mutant strain. The mutant strain of the present invention can be grown in a medium containing both methanol and glucose, with the effect that the growth of the strain and production of a heterologous protein proceed at the same time. Accordingly, a heterologous protein can be produced in a large amount in a short time.

Mutant strain of Pichia pastoris which utilizes methanol in the presence

A mutant AOX2 (alcohol oxidase 2) promoter derived from the natural AOX2 promoter by base sequence deletion, insertion, or substitution is disclosed. A microbial strain carrying a gene under the control of such a mutant AOX2 promoter can be obtained by growing in a methanol-containing medium a strain incapable of producing AOX encoded by the AOX1 gene but carrying the AOX2 gene under the control of the natural AOX2 promoter. A heterologous protein may be produced by cultivating the mutant strain with the desired heterologous protein gene incorporated downstream from the mutant AOX2 promoter. A plasmid carrying the mutant AOX2 promoter is also provided.

Mutant AOX2 promoter, microorganism carrying same, method of preparation

A method for preparing foreign protein in yeast using an expression recombinant DNA comprising DNA encoding the serum albumin signal peptide adjacent to DNA encoding the foreign protein is disclosed.

Method of preparing foreign protein in yeast, recombinant DNA,

Novel signal peptides capable of functioning in yeasts have the amino acid sequence:

DNA's encoding signal peptides

A human prourokinase mutant in which the entire or a partial epidermal growth factor domain of human prourokinase is deleted or a partial epidermal growth factor domain of human prourokinase is replaced by one or more different amino acid residues, said mutant having a longer blood half-life than naturally occurring human prourokinase while retaining prourokinase enzymatic activity. In this human prourokinase mutant the region selected from the group consisting of: (a) from asparagine (10) to cysteine (42); (b) from asparagine (10) to aspartic acid (45); and (c) from asparagine (10) to threonine (49) is missing.

Human prourokinase mutants

A DNA sequence of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) promoter and a process for preparing a heterologous protein utilizing said DNA sequence as a promoter are disclosed, said GAP-DH promoter comprising a region upstream of an initiator codon of the GAP-DH protein up to -164 bp as a minimum unit. Since the GAP-DH promoter is small in size, a physiologically active substance can be expressed in yeasts effectively and recombination of DNA can be simplified.

Yeast promoter truncated GAP-DH

A method of producing a heterologous protein in yeast is disclosed, which comprises transforming a yeast Saccharomyces cerevisiae with recombinant DNA comprising a promoter selected from the group consisting of a yeast promoter or a hybrid promoter derived from a yeast promoter, particularly a hybrid promoter containing the enhancer region of SV40 virus, and a gene coding for a heterologous protein such as HBsAg and Pre S-HBsAg, particularly full length Pre S-HBsAg culturing, the resulting transformed cells to express the gene coding the heterologous protein, and isolating the heterologous protein from the cultured medium. GAP-DH and PH05 promoters and miniaturized ones can be used as the yeast promoter. The expression of the full length Pre S-HBsAg is carried out in the same expression system as that for 2nd or 3rd Pre S-ABsAg.

Yeast promoter and process for preparing heterologous protein

A recombinant plasmid wherein at least two DNA fragments containing a Baker gene and a Charlie gene of the yeast 2 .mu.m circular plasmid are incorporated into a plasmid containing genes encoding a physiologically active substance is disclosed. Also, a strain transformed by the recombinant plasmid is disclosed.

Recombinant plasmid and microbial strain transformed by said plasmid

Growing community of inventors

Osaka, Japan

Haruhide Kawabe