Secretory expression of human ScFv against keratin in Pichia pastoris and its effects on cultured keratinocytes

Abstract

We have got the humanized antibody with high affinity and specificity against keratin by phage antibody library technology.
To improve protein yields and get high affinity and specific anti-keratin antibody, we chose to express it in Pichia pastoris. Anti-keratin ScFv gene from plasmid p3MH/ScFv was subcloned into vector pPIC9K. After confirmed by DNA sequence analysis,
the recombinant plasmid pPIC9K/ScFv was transduced into the genome of GS115 P. pastoris. Muts multiple insert transformants were screened by G418 and induced by 5mL/L methanol to express soluble ScFv. After 6days
of methanol induction, anti-keratin ScFv was efficiently secreted into the medium. Western blot and ELISA assay proved the
expressed protein had specific keratin-binding activity. After purification, we examined its effect on cultured keratinocytes
by Cell cycle analysis, Which indicated that human ScFv against keratin 17 can inhibit the proliferation of keratinocytes
by influence the synthesize of keratinocyte DNA. The successful expression of anti-keratin ScFv in P. pastoris laid a solid foundation for its further application.