Strategies to maximize expression of rightly processed human interferon alpha2b in Pichia pastoris.

The human interferon alpha 2b (IFN alpha2b) belongs to the interferon family of cytokines that exerts many biological functions like inhibition of virus multiplication, repression of tumour growth and other immunological functions. Herein, a synthetic gene coding for human IFN alpha2b was cloned and integrated into a methylotropic yeast-Pichiapastoris. The recombinant human IFN alpha2b protein (approximately 19kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300mg/L with nearly 93% recovery on purification using a single anion exchange chromatography step. A novel media component dimethyl sulphoxide (DMSO) was found to aid in expression of rightly processed IFN alpha2b form with dramatic reduction in the expression of a 20kDa IFN isoform contaminant frequently observed by other workers. The identity of the 20kDa isoform was confirmed by N terminal sequencing which showed extra eleven amino acids at the N terminal portion of the IFN molecule obtained due to incorrect processing by the host KEX2 protease. The purified IFN alpha2b (19kDa) preparation was confirmed by N terminal sequencing, and characterized by MALDI-TOF and Agilent 2100 Bioanalyzer. The bioassay of the recombinant protein gave a specific activity of >2x10(8)IU/mg.

Yeast extract mediated autoinduction of lacUV5 promoter: an insight.

We report a simple and cost-effective autoinducible media component responsible for the autoinduction of proteins in Escherichia coli under lacUV5 promoter system. Yeast extract (YE) at high concentration was found to stimulate the expression of T7 RNA polymerase in BL21(DE3) cells while such an effect was not seen in BL21A1 cells. A systematic study on the effect of varying concentrations of YE indicated several folds higher expression of genes viz., human granulocyte colony stimulating factor (rhGCSF), human interferon alpha 2b (rhIFN-alpha2b) and Staphylokinase (rSAK) in BL21(DE3) cells in the absence of any specific inducer like IPTG or additional lactose. Additional investigations on the inducible component of the YE revealed the presence of significant amount of endogenous lactose as the contributory factor for the observed autoinduction phenomenon. This paper highlights the easy scalability of the use of the present media component for large-scale production in biotechnology industry.

Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia pastoris using Agilent 2100 Bioanalyzer.

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.

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris.

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.

High yielding recombinant Staphylokinase in bacterial expression system--cloning, expression, purification and activity studies.

Staphylokinase (SAK) is emerging as an important thrombolytic agent. In this report, we describe the cloning, expression, purification and activity studies of the SAK gene of Staphylococcus aureus from a custom synthesised SAK gene. The SAK gene of 411 bp yielded a protein of approximately 15 kDa when expressed under pET21a vector using IPTG as an inducer in BL21 (DE3) pLysE codon Plus cells. The recombinant SAK (rSAK) was soluble in nature and constituted nearly 35% of the total cellular protein as estimated by densitometry scanning. Fermentation studies were carried out to optimize various parameters for maximizing the yield of rSAK and with the optimized medium, the yield of rSAK was nearly 2.8 g/L of fermentation broth, which is highest yield of rSAK expressed in any bacterial system till date. Two simple purification steps of ion-exchange chromatography yielded homogenous rSAK with almost 36% recovery. The purified SAK protein was characterized by MALDI-TOF and by plasminogen activation studies. The rSAK was found to be active by the chromogenic substrate assay method.