ABSTRACT
Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment
ABSTRACT
Objective To study the expression, purification and bioactivity of a recombinant fusion protein consisting of anti-HBs single chain Fv and interleukin-2. Methods The engineering bacterium M15[pQE-ScFv-IL-2] which can express the fusion protein consisting of anti-HBsAg single chain Fv and interleukin-2, was induced by IPTG, then a 43kD recombinant protein was identified by SDS-PAGE and Western-blot analysis. Results The ratio of target protein to total protein of host reached 18%. Further analysis confirmed that the recombinant protein formed inclusion body in the cytoplasm of bacteria. 95% purity could be achieved after two-step purification of ScFv-IL-2, including Ni metal chelating chromatography (the first) and ion-exchange chromatogram (the second). The bioactivity assay of the purified product showed that the antibody-cytokine fusion protein could bind to HBsAg specifically and stimulate the proliferation of CTLL-2. Conclusion These results suggest that the fusion protein retains the bioactivity of its parental molecules, and may be a potential gene-engineering targeting drug for the treatment of chronic hepatitis B and other relevant diseases.
ABSTRACT
Objective The aim of the study was to develop a purification procedure on Pichia pastoris GS115/Fab expressing human anti-HBsAg Fab fragment. Methods Purity and yield ratio and conjugated activity of purified Fab fragment were analyzed with three purified ways of goat anti human Fab affinity chromatography and 14F7 monoclonal antibody affinity column, as well as Ion exchange Size exclusion column. Results 98% purity was reached through 14F7 monoclonal antibody column, and 95% purity was gained after goat anti human Fab fragment column. But yield ratio of the two affinity columns was low, being 35% and 55%, respectively. ForIon exchange Size exclusion column, purity and yield ratio of Fab fragment were very good, being 93.8% and 80% or more, respectively. Results of ELISA analysis showed that purified Fab fragment through three columns could bind to HBsAg specifically. Conclusion The purification process of recombinant anti-HBsAg Fab fragment was established. It lays a foundation for industrialization and clinical research of human anti-HBsAg Fab fragment.