Construction, expression and characterization of a fusion protein HBscFv-IFNγ in Komagatella (Pichia) pastoris X33.

HBscFv-IFNγ, a fusion protein constructed by fusing γ-interferon (IFNγ) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNγ, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNγ were connected into HBscFv-IFNγ by the linker (Gly4 Ser)3 , and then the multicopy recombinant plasmids pPICZαA/(HBscFv-IFNγ)1,2,4 were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNγ, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNγ displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNγ in a purity of 95-98%. The HBscFv-IFNγ was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNγ has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNγ in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.