title: A system for dual protein expression in Pichia pastoris and Escherichia coli.
abstract: We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.
authors.affiliation: Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Lueking
authors.forename: C
authors.initials: C
authors.lastname: Holz
authors.forename: C
authors.initials: C
authors.lastname: Gotthold
authors.forename: H
authors.initials: H
authors.lastname: Lehrach
authors.forename: D
authors.initials: D
authors.lastname: Cahill
mesh_terms: D001923:Brain Chemistry; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D010843:Pichia / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D018076:DNA, Complementary