title: [Enhancement of human insulin precursor production by increasing the copy number in Pichia pastoris].
abstract: OBJECTIVE
  The aim of this study was to further increase the yield of insulin precursor by Pichia pastoris.
  
  
  METHODS
  For this, we transformed the expression vector pPICZalpha-IP into P. pastoris X-33 using electroporation and screened two mutant strains B4 and S6 on the YPD plate containing 100 microg/mL zeocin. Both could overexpress human insulin precursor. Taking B4 and S6 as start strains, we repeatedly transformed SacI linearizing pPICZalpha-IP into P. pastoris X-33 by electroporation, then screened a new mutant strain 2B4 (with 7 copies) on the 1000 microg/mL zeocin YPD plate.
  
  
  RESULTS
  After cultivation, the human insulin precursor yield of 2B4 strain was 2.7 fold higher than that of B7. Meanwhile, the cell growth was not inhibited. The target gene transcription level of 2B4 was 2 fold higher than that of B7 by real-time quantification PCR.
  
  
  CONCLUSIONS
  The strategy of combining resistance screening and repeated electroporation was efficient to increase the copy number of target gene, so as to facilitate higher transcription level and enhance objective recombinant protein yield.
authors.affiliation: Wuxi Medical School, Jiangnan University, Wuxi 214122, China. gong.xiangyi@163.com
authors.forename: Xiangyi
authors.initials: X
authors.lastname: Gong
authors.forename: Chongyang
authors.initials: C
authors.lastname: Ding
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D003001:Cloning, Molecular; D018628:Gene Dosage; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D011994:Recombinant Proteins