Research Corporation Technologies, Inc.

Phillips Petroleum Company

Kathryn A Barr

Gregory P Thill

Michael Miller Harpold

David W Stroman

Juerg F Tschopp

Frank H Gaertner

Mary J Hagenson

Kotikanyadan Sreekrishna

Russell A Brierley

William D Prevatt

Ronald D Klein

Geneva R Davis

Patricia Koutz

Kotikanyadanam Sreekrishna

Sharon A Hopkins

Kotikanyad Sreekrishna

Mary Jane Hagenson

A method for producing one or more Bacillus toxin polypeptides by culturing methylotrophic yeast cells which have a gene(s) capable of expressing the Bacillus toxin polypeptide(s) in such cells under conditions that the gene(s) is/are transcribed is provided. The toxin polypeptide encoding segment of the gene(s) has a G+C content of about 40%-55%, and preferably comprises methylotrophic yeast codons. The preferred species of yeast for expressing such synthetic Bacillus toxin gene(s) is Pichia pastoris. Bacillus toxin polypeptides encoded by synthetic genes are expressed at high levels in transformed methylotrophic yeast cells. The toxin expressing cells may be administered as live cells or heat-killed whole cells to provide an insecticidal composition for killing susceptible insect larvae. Also provided by the present invention are DNAs capable of transforming methylotrophic yeast to express one or more Bacillus toxin polypeptides, cultures of such yeast cells transformed with such DNAs and novel Bacillus toxin polypeptides made by the method of the invention. The transformed yeast cells of the present invention are readily ingested as food by insect larvae which are susceptible to the toxin polypeptides.

Production of Bacillus entomotoxins in methylotrophic yeast

The present invention is directed to expression cassettes comprising a 5' regulatory region from at least one of the Pichia pastoris AOXI gene, p40 gene, DASI gene or HIS4 gene, operably linked to an HSA structural gene including the HSA signal sequence. The HSA structural gene has a translational start codon within 0 to 11 deoxyribonucleotides from the 5' end of the HSA structural gene and is operably linked to a 3' termination sequence. Further, the adenine and thymine content of the intervening deoxyribonucleotides is in the range from about 55 to about 64%. The expression cassette may be an autonomously replicating vector or an integrative vector. Other embodiments of the present invention include Pichia pastoris strains transformed with the HSA expression cassettes and processes for secretion of HSA using the expression cassettes.

Expression of human serum albumin in Pichia pastoris

Two novel linear DNA plasmids are described. Also, novel fragments of the plasmids containing the autonomous replication sequence (ARS), and thus capable of self-maintenance as extra chromosomal elements are provided. These novel DNA sequences of the present invention are useful in a vector for self-maintenance in Pichia pastoris and are also useful as plasmid vectors which can be shuttled between yeast and bacteria.

Pichia pastoris linear plasmids and DNA fragments thereof

Growing community of inventors

Bartlesville, OK, United States of America

Kathryn A Barr