title: [Expression of anti-keratin human Fab in Pichia pastoris and optimization of expression condition].
abstract: 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.
authors.affiliation: Institute of Dermatology and Venerology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. fanjianyongfmmu@sina.com
authors.forename: Jian-Yong
authors.initials: JY
authors.lastname: Fan
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Zhu
authors.initials: Z
authors.lastname: Shen
authors.forename: Cheng-Xin
authors.initials: CX
authors.lastname: Li
authors.forename: Tian-Wen
authors.initials: TW
authors.lastname: Gao
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Liu
mesh_terms: 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
chemical_list: D000906:Antibodies; D004271:DNA, Fungal; D007140:Immunoglobulin Fab Fragments; D007633:Keratins; D000432:Methanol