Phillips Petroleum Company

Research Corporation Technologies, Inc.

Kotikanyadan Sreekrishna

Motohiro Fuke

Juerg F Tschopp

Gregory P Thill

William D Prevatt

Russell A Brierley

Kathryn A Barr

Rica H Potenz

Frances M Davis

Farhad Marashi

Ricky N Wong

Karen M Vineyard

Kathryn Parker

Robert D Barr

W Richard Mccombie

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

A process for the production of HSA in Pichia pastoris cells comprising cultivating Pichia pastoris cells capable of expressing HSA at a pH of about 5.7 to about 6.4 contemporaneously with the expression of HSA.

Novel DNA constructs comprising yeast regulatory regions plus the structural coding region for human tumor necrosis factor, are disclosed. These novel constructs are incorporated into a variety of linear and circular plasmids. Such plasmids are used for yeast transformation and ultimately for the production of human tumor necrosis factor by yeast.

Yeast production of human tumor necrosis factor

Yeast strains are transformed with, and selected for, using DNA fragments encoding a gene function not present in the wild-type yeast strains. The invention requires transforming the yeast with DNA fragments which comprise the gene function which the wild-type yeast strain is lacking; regeneration of the transformants on a non-selective regeneration medium; then selection of those colonies which are capable of growth on a carbon and energy source which requires expression of the gene function provided by the DNA fragment. Thus, transformation with such DNA fragments allows for positive selection of transformed strains.

Carbon and energy source markers for transformation of strains of the

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Bartlesville, OK, United States of America

Kotikanyadan Sreekrishna