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 pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, 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.

Cassette series designed for live‐cell imaging of proteins and high‐resolution techniques in yeast

“…Additionally, reverse primers recommended for previously reported pFA6a-based plasmids (Funakoshi and Hochstrasser, 2009; Janke, et al, 2004; Petracek and Longtine, 2002); Sung and Huh, 2007; Tagwerker et al, 2006; Wach et al, 1997) are compatible with this new series of cassettes, while forward and reverse primers designed for pKT modules (Sheff and Thorn, 2004) are identical. Plasmids including the kanMX and hphMX4 genes are applicable for C-terminal tagging in the fission yeast Schizosaccharomyces pombe (Bahler, et al, 1998; Marti, et al, 2003) while kanMX and the zeocin resistance gene can be used in Pichia pastoris (Papakonstantinou, et al, 2009). Our designs can be implemented similarly to a described system for C-terminal epitope switching (Sung, et al, 2008) as well as to target gene disruptions and deletions in yeast.…”

“…We also utilized for the first time a photoswitchable green-to-red variant, mEos2, in yeast while analyzing its expression and fluorescent properties. Furthermore, we have increased the repertoire of selection markers in S. cerevisiae by incorporating the Streptoalloteichus hindustanus bleomycin ( Sh ble ) gene that confers resistance to Zeocin™, as previously published (Frazer and O’Keefe, 2007; Papakonstantinou, et al, 2009). Each cassette is available with a kanMX or hphMX4 gene, in addition to the following selective marker genes TRP1, LEU2 , or URA3 used with appropriate auxotrophic yeast strains while maintaining primer homology consistent with established cassettes derived from pFA6a.…”

Hygromycin-resistance vectors for gene expression inPichia pastoris

“…GFP can be expressed in P . pastoris (Lin‐Cereghino et al ., ; Papakonstantinou et al ., ; Yang et al ., ). In this work, GFP expression from the hygromycin‐resistance vector was similar to expression from a Zeocin‐resistance vector or expression as an unmarked knock‐in gene (Yang et al ., ).…”

“…For the GSH2 expression vector, a G418/Kan resistance vector was constructed as described previously (Papakonstantinou et al ., ). A fragment containing the G418/Kan resistance gene was amplified from pUG6 (Guldener et al ., ), using the primer pair kanMXup/Ttef_dn (Table ), and used to replace the Nco I– Eco RV region containing the Zeo R gene of pGAPZB, yielding the expression vector pGKB.…”

“…Currently, the dominant antibiotic makers that are available for P . pastoris are limited to the Sh ble gene from Streptoalloteichus hindustanus (Zeocin resistance) (Drocourt et al ., ), the blasticidin S‐deaminase gene from Aspergillus tererus (blasticidin resistance) (Kimura et al ., ), the nourseothricin acetyltransferase gene from Streptomyces noursei (nourseothricin resistance) (Chen et al ., ) and the kanamycin‐resistance gene of transposon Tn903 (G418 resistance) (Lin‐Cereghino et al ., ; Papakonstantinou et al ., ; Scorer et al ., ).…”