Stability of Recombinant Human Interferon Alpha-2b Produced In Methylotropic Yeast Pichia Pastoris

The stabiIity of recombinant human interferon aIpha -2b (rhlfNa-2b) remains a great challenge for PharmaceuticaI science. In previous research we constructed open reading frame encoding rhlFNa-2b and produced the protein in Pichia pastoris {P. pastoris). This research was aimed to study the stabilityof rhlFNa-2b in threeparameters: temperature, pH and shelf life. The rhlFNa-2b was overproduced by using buffered methanol complex medium (BMMY) at 30 °C for 48 h with 2% of methanol as inducer. Filtration of protein was used by mini mate™ tangential flow filtration system with molecular weight cut off {MWCO) 5 kDa. Purification of rhlFNa-2b was performed by immobilized affinity chromatography column using AKTA purifier system. Colorimetric bicinchoninic acid as say informed that the yield of purified rhlFNa-2b was 10.92 mg/L (OD600 = 2.3). Sodium dodecyl sulphatepolyacrylami de gel electrophoresis {SOS-PAGE) and Western Blot a na I yses confirmed that the protein was rhl FNa-2b with 24 kDa in size. Matrix assisted laser desorption ionization-time offlight/time of flight (MALDI-TOF/TOF) mass spectrometry identified the protein as hlFNa-2b with 22% of amino acid coverage. Non reducing SDS-PAGE and Image J software analyses showed that temperature incremeot, acidic and basic pH as well as shelf life length caused protein aggregation and degradation. 3-[4.5-dimethylthiazol-2il)-2.5-dipheoyltetrazolium bromide (MTT) assay informed that the aggregation and degradation reduced the anti-proliferative activity of rhlFNa-2b on human breast cancer MCF-7 cell line. To conclude,all parameters give an impact on rhlFNa-2b stability with the most influencing parameter was temperature at 25 •c. These data can be used to develop rhlFNa-2b formulations as therapeutic protein. Int.J.Res.Pharm.Sci, Vol.6 No.4, Hal.312-320