A system for dual protein expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expressio n vector for the production of recombinant proteins in both host systems. I n this vector, an E. coli T7 promoter region, including the ribosome bindin g site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detect ion and purification of the target protein, the vector contains an amino-te rminal oligohistidine domain (His6) followed by the hemaglutinine epitope ( HA) adjacent to the cloning sites. AP. pastoris autonomous replicating sequ ence (PARS) was integrated enabling simple propagation and recovery of plas mids from yeast and bacteria (1). In the present study, the expression of h uman proteins in P. pastoris and E. coli was compared using this single exp ression vector. For this purpose we have subcloned a cDNA expression librar y deriving from human fetal brain (2) into our dual expression T7 vector an d investigated 96 randomly picked clones. After sequencing, 29 clones in th e correct reading frame have been identified, their plasmids isolated and s huttled from yeast to bacteria. All proteins were expressed soluble in P. p astoris, whereas in E. coli only 31% could be purified under native conditi ons. Our data indicates that this dual expression vector allows the economi c expression and purification of proteins in different hosts without subclo ning. (C) 2000 Academic Press.