Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia...

Recombinant human granulocyte colony stimulating factor (rhGCSF) was expressed in methylotrophic yeast Pichia pastoris under the control of AOX1 promoter after integration of the GCSF gene into P. pastoris genome. Methanol induction of the Pichia integrants yielded only 2mgl(-1) of rhGCSF whereas inclusion of surfactants during induction enhanced the yields to the level of 200-250mgl(-1) in shake flask studies after 72h of induction. Preliminary studies in a bioreactor showed rhGCSF expression levels of 6mg rhGCSF g(-1) methanol day(-1) which is significantly higher to the reported value of 0.4mg rhGCSF g(-1) methanol day(-1) reported till date for Pichia derived rhGCSF. A single step purification protocol of shake flask derived rhGCSF yielded homogenous rhGCSF protein of >99% purity. Even though, purified rhGCSF showed a single band on reducing SDS-PAGE, examination of the same protein on Agilent 2100 Bioanalyzer, revealed two closely unresolved peaks. Such a pattern was also observed for crude rhGCSF preparations. Mutagenesis of the O-glycosylation site of rhGCSF (Thr(133) to Leu(133)) showed a single peak on bioanalyzer, which overlapped with the peak obtained for a non-glycosylated rhGCSF. Our data discloses for the first time the novel use of Agilent Bioanalyzer to detect glycoforms of proteins in crude and purified preparations and such a tool could be easily applied for glycoprotein profiling of monoclonal antibodies and other fusion proteins expressed in mammalian cells. This is the first report of a simple, rapid, sensitive and a cost-efficient tool for detection of glycoproteins.