title: Expression of GFP using Pichia pastoris vectors with zeocin or G-418 sulphate as the primary selectable marker.
abstract: Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6alpha series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZalphaA, B and C vectors with the Tn903 kan(R) marker from pFA6a KanMX6, which confers G-418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa-histidine-tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production.
authors.affiliation: ARC Special Research Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Theo
authors.initials: T
authors.lastname: Papakonstantinou
authors.forename: Simon
authors.initials: S
authors.lastname: Harris
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D025141:Drug Resistance, Fungal; D005818:Genetic Engineering; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010843:Pichia / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic
chemical_list: D000890:Anti-Infective Agents; D000903:Antibiotics, Antineoplastic; D005839:Gentamicins; D011994:Recombinant Proteins; D001761:Bleomycin; D049452:Green Fluorescent Proteins; C105427:Zeocin; C010680:antibiotic G 418