Expression Of Vitreoscilla Hemoglobin Improves Cell Density And TNFRⅡ-FC Yield In A Recombinant Pichia Pastoris

TNFRⅡ-FC (trade name:ENBREL) is a protein comprised of the extracellular domains of TNF (tumor necrosis factor) receptorⅡattached to a portion of an IgG immunoglobulin.It acts primarily to bind and neutralize TNF,thus blocks the TNF mediated abnormal immune response and inflammatory response. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons.enbrel is produced by mammalian cell expression system.and first approved for reducing signs and symptoms in patients with moderately to severely active rheumatoid arthritis (RA) in 1998.The high cost and low yield of this system is still the bottle neck of large scale production and application of this kinds of drug.The Pichia pastoris is widely used as an expression host to express various recombinant proteins because of its potential for high expression levels, efficient secretion of extracellular protein, posttranslational modifications and growth to high cell densities on defined minimal medium.Due to the differences in N-glycosylation phthway between Pichia pastoris and mammalian cell lines,The high-mannose glycan of Pichia pastoris limited the application of Pichia pastoris to produce glycoproteins.with the great advancements in Engineered N-glycosylation phthway use Pichia pastoris as host to produce recombinant glycoproteins becomes possible.Vitreoscilla hemoglobin(VHb)is the first described bacterial hemoglobin (Wakabayashi et al. 1986) and is the best characterized of prokaryotic origin.It has been proposed that VHb binds oxygen and transfers it to the terminal oxidases (Park et al. 2002). Therefore, expression of the VHb gene (vgb) could enhance host growth and metabolism under oxygen limited conditions Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous bacterial hosts to enhance cell density, oxidative metabolism, and protein production, especially under O2-limiting conditions (Stark et al. 1999; Erenler et al. 2004).First,The TNFRⅡ-FC DNA sequence was inserted into pichia pastoris vector ppiczαA to form a new expression vector.then,the vector was integrated into the pichia pastoris and the expression was confirmed by SDS-PAGE and Western blot. The confirmed transformants were denoted as GE. The protein expressed was purified by affinity chromatography and immune activity was measured.The fragment of ppic9 containing Amp resistance gene was inserted into ppiczαA digested with the same enzymes and the VHb sequence was inserted into this vector to form a expression vector.the vector was integrated into the pichia pastoris strain GE the new engineered strain was denoted as GE-VHB.Cell growth was monitored by optical density at 600 nm Wavelength (OD600),.TNFRⅡ-FC contents were determined by Western blot. Bands were scanned and quantified by Bandscan. the results showed that VHB enhances cell density and TNFR-FC yields in both O2-limiting and non-limiting conditions, especially in O2-limiting condition.In this study,we used pichia pastoris as host to expressing TNFRⅡ-FC ,which can be a reference for the future expression of this kinds of drug. Then,the VHb sequence was integrated into the pichia pastoris expressing TNFRⅡ-FC.The results showed thatVHB enhances cell density and TNFR-FC yields in both O2-limiting and non-limiting conditions, especially in O2-limiting condition.which offer a effective approach to solve the O2-limiting in fermentation.