Biotechnology Bulletin ›› 2015, Vol. 31 ›› Issue (9): 244-250.doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.035

• Research report • Previous Articles    

Expression, Purification and Bioactivity of Recombinant Human Interleukin-1α Expressed in Pichia pastoris

Li Bin, Xu Xiaoya, Yang Ganggang, Cui Qingqing, Yang Yajuan, Xu Cunshuan   

  1. (State Key Laboratory Cultivation Base for Cell Differentiation Regulation and Henan Bioengineering Key Laboratory,Henan Normal University,College of Life Science,Henan Normal University,Xinxiang 453007)
  • Received:2014-12-22 Online:2015-09-15 Published:2015-09-16

Abstract: This work is to secret and express human interleukin-1α(rhIL-1α)in Pichia pastoris and optimize the fermentation and purification process of rhIL-1α for obtaining the rhIL-1α with high-purity, high-expression and owing biological activity. The gene hIL-1α amplified by PCR was constructed into the eukaryotic expression vector pPICZαA/hIL-1α, and then it was transformed into the P. pastoris X-33 strain via electroporation.The engineering strain with high-expression of rhIL-1α was screened and assayed by the methods of PCR and SDS-PAGE, further indentified by Western blot. The expressed product was purified by the DEAE Sepharose Fast Flow ion exchange chromatography, and the bioactivity of it to human cancer Bel-7402 cell was initially assayed. Results showed that inducing rhIL-1α by methanol for 4 d at shaking flask level, the expression reached 30mg/L, the test by Western blot revealed that specific binding activity of rhIL-1α was detected;the purity of the rhIL-1α reached about 95% and the yield was about 40%;rhIL-1α inhibited the proliferation of Bel-7402 cells. In conclusion, recombinant engineering vector of rhIL-1α was successfully constructed, and it was highly expressed in P. pastoris, which lays groundwork for further study of its function and bioactivity.

Key words: recombinant human interleukin-1α, Pichia pastoris, expression, purification, Bel-7402 cell