Source Anti-HBsAg ScFv In E. Coli And Pichia Yeast Expression, Purification And Characterization Of Recombinant Human

Hepatitis B virus infection is a worldwide public health problem affecting about 350 million people. Monoclonal antibodies or their fragments are only useful in prevention if they bind to and block surface regions of the virus which are essential for the infection of hepatocytes. The objective of this paper is to generate a single-chain Fv antibody against HBsAg (HBscFv) from a human Fab fragment screened in previous study, and to express the HBscFv in Escherichia coli and Pichia pastoris, and to purify and characterize the recombinant HBscFv. Generated by gene splicing by overlap extension, HBscFv was sequenced and sub-cloned into E. coli expression vector pQE-40. The recombinant plasmid pQE-HBscFv was then transformed into E. coli M15[pREP4]. Induced with IPTG, recombinant HBscFv was expressed insolubly and at high level in the bacteria cytoplasm, approximately 31.2% of the total protein of the recombinant E. coli M15[pQE-HBscFv]. Furthermore, the recombinant HBscFv was also found to be insoluble in E. coli Origami (DE3), a mutant lacking thioredoxin reductase and glutathione reductase in favor of disulfide bonds formation. Refolded by a simply way, the inclusion body of HBscFv was found having specific binding to HBsAg. To purify and refold the inclusion body of HBscFv, the recombinant E. coli M15[pQE-HBscFv] was cultured and induced in an optimum condition. The resulting inclusion body was solubilized and purified by IMAC, IEX, IEX/IMAC and IMAC/GF. It was found that one-column purification of the inclusion body by IMAC resulted HBscFv in 85% purity, and if it was polished by GF, HBscFv in 97% purity could be obtained. The purified HBscFv was then refolded by 3 methods. It was found that dialysis was efficient than dilution or in situ refolding, and dialysis-against-GuHCl was efficient than dialysis-against-urea. Under the optimum condition, the recovery of protein and the relative specific bioactivity were (61.08±1.45)% and (2.66±0.13)A450/630 U/μg, respectively. The final yield of active HBscFv was 34.7mg per liter flask-shaking culture. The bioactivity and physical character of recombinant HBscFv was determined. The affinity constant of HBscFv was confirmed to be (2.30±0.23)×107 M-1 by non-competitive ELISA. The neutralizing activity of HBscFv was proved in vivo in transgenic mouse with a titer of 31.58 U/mg. The mass weight of HBscFv was measured to be 27314Da by MALDI-TOF MS, with a deviation of 8Da. The peptide-map of HBscFv was analyzed by MALDI-TOF MS and more than 60% fragments recognized by trypsin could be found in the MS peak, including the sequent 5 fragments in the N-terminal and a fragment with disulphide bond. The heterogeneity of pI was found when the pI was measured by isoelectric focusing. The second and 3D structures of HBscFv were predicted by PHDsec and homologous-modeling, respectively, and it was found that there were 9 sheets in VH domain and 8 sheets in VL domain of HBscFv. Secretory native HBscFv was produced in P. pastoris. Amplified from plasmid pGEM-HBscFv, native HBscFv was sub-cloned into expression vector pPICZalphaA. The resulted recombinant plasmid was linearized and transformed into P. pastoris GS115 or KM71 by the methods of LiCl or spheroplast transformation. It was found that 75% Pichia transformants had integrated target gene, and 3%~8% transformants could growth in the medium containing 2000mg/L Zeocin. The transformants resistant to 100mg/L, 1000mg/L and 2000mg/L Zeocin were cultured and induced with methanol and HBscFv-like protein could be found in the culture supernatant. The amount of HBscFv-like protein produced by 1000mg/L and 2000mg/L Zeocin-resistant transformants was similar, but it was significant higher than that produced by 100mg/L Zeocin-resistant transformants. The 32kD protein in the culture supernatant was confirmed to be HBscFv by Western-blot. Quantitative analysis showed that HBscFv produced by yeast was in a level of 28% of the total protein of the culture supernatant, with a yield of 40mg/L. HBscFv-like protein can't be found in yeast cell, suggesting that HBscFv can be secreted properly under the lead of α-MF signal peptide. The native HBscFv expressed by P. pastoris was purified and characterized. Fractionated by 50% saturation of ammonium sulfate, the culture supernatant waspurified by IEX and immuno-affinity-chromatography, resulting recombinant HBscFv with 95% purity. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached the peak in 72h and then decreased in the late of induction. The epitope difference between E. coli and P. pastoris-produced HBscFv was analyzed with mouse anti-HBscFv McAb. It was found that the binding intensity of P. pastoris-produced HBscFv to the McAb was weak than that of E. coli-produced HBscFv. PAS glycoprotein staining suggests that HBscFv produced by yeast is lowglycosylated or not glycosylated protein. In conclusion, the gene of a human scFv against HBsAg was constructed and efficiently expressed in E. coli and P. pastoris. The expression of an antibody fragment against HBsAg in P. pastoris was reported for the first time, moreover, the neutralization bioactivity of this category of antibody was proved in vivo for the first time. This would certainly shine lights on the study of the mechanism of this scFv binding its antigen, and on the construction of double or multi-specific antibodies those could be applied to preventing, diagnosing and treating of diseases associated with HBV.