[Expression of anti-keratin human Fab in Pichia pastoris and optimization of expression condition].

Aim: To express human anti-keratin Fab in Pichia pastoris secretively and optimize the expression condition.
Methods: Genes of kappa chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/Fab were subcloned into vectors pPIC9K and pPICZalphaA respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/L and pPICZalphaA/Fd were transducted into the genome of GS115 Pichia pastoris using two-step integrating technology. Mut(+) multiple insert transformants were screened by G418 and Zeocin. The pH value and methanol concentration was adjusted to optimize the expression condition.
Results: Under the optimized expression condition, the Fab of anti-keratin antibody was efficiently secreted into the medium. Western blot assay proved that the expressed protein had specific keratin binding activity.
Conclusion: The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.