title: High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.
abstract: The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK. CemalGurkan@cal.berkeley.edu
authors.forename: Cemal
authors.initials: C
authors.lastname: Gurkan
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Symeonides
authors.forename: David J
authors.initials: DJ
authors.lastname: Ellar
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000429:Alcohol Oxidoreductases; D003470:Culture Media; D007128:Immunoglobulin Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C019643:alcohol oxidase