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


CONCLUSIONS
The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.
Fan
Jian-Yong
JY
Institute of Dermatology and Venerology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. fanjianyongfmmu@sina.com
Wang
Gang
G
Shen
Zhu
Z
Li
Cheng-Xin
CX
Gao
Tian-Wen
TW
Liu
Yu-Feng
YF
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D007633:Keratins / Q000276:immunology*; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
D000906:Antibodies; D004271:DNA, Fungal; D007140:Immunoglobulin Fab Fragments; D007633:Keratins; D000432:Methanol