New Strategy for Expression of Recombinant Human Prolyl-4-Hydroxylase in Pichia pastoris
New Strategy for Expression of Recombinant Human Prolyl-4-Hydroxylase in Pichia pastoris
Yating Cheng1, Wenlong Shi1, Xue Xiao2, Qirong Zhang2, Qihao Zhang1, Zhijian Su1, Qi Xiang1,2* and Yadong Huang1,2
Schematic diagram of the pPICZαA-P4Hα2 and PHIL-PDI constructs. (a) Construction of the expression plasmid pPICZαA-P4Hα2. (b) Construction of the expression plasmid PHIL-PDI. (c) Nucleic acid electrophoresis of recombinant plasmid pPICZαA-P4Hα2 and PHIL-PDI. M: DNA Ladder 10000; lane 1: the monoclones of recombinant plasmid pPICZαA-P4Hα2; lane 2: the monoclones of recombinant plasmid PHIL-PDI.
Analysis of the expression of rhP4H in P. pastoris GS115. The apparent molecular mass of rhP4H is ~60 kDa. (a) Detection of P4Hα2 and PDI at the mRNA level at different concentrations of Zecion. (b) SDS-PAGE analysis of the expression of rhP4H selected by 0.1, 0.5, 1.5, 2.5 and 3.5 mg/mL Zecion, respectively. M: middle molecular weight protein markers. (c) Western blotting analysis of PDI and P4Hα2 expression at different concentrations of Zecion. (d) SDS-PAGE analysis of the expression of rhP4H induced by 0.5, 1.0, 1.5, 2.0% methanol, respectively. M: middle molecular weight protein markers. (e) Western blotting analysis of the expression of PDI and P4Hα2 at different concentrations of methanol.
SDS-PAGE analysis of the expression of rhP4H. rhP4H is present in the various ammonium sulfate fractions. M: middle molecular weight protein markers; lane 1: supernatant sample of rhP4H after induction; lanes 2–7: reconstituted sample of rhP4H after precipitation with 20%, 30%, 40%, 50%, 60% and 70% ammonium sulfate, respectively.
Activity spectrum of rhP4H with Dansyl-Gly-Phe-Pro-Gly-OEt as the substrate in supernatant of pastoris extract or in fermentation broth of yeast cells. (a) Blank control. (b) Substrate and product standard. (c)The activity identification mass spectrum of rhP4H. (d) The activity identification mass spectrum of PDI.
Activity spectrum of rhP4H with Pal-Gly-Gln-Pro-Arg as the substrate in supernatant of pastoris extract or in fermentation broth of yeast cells. (a) Blank control. (b) The activity identification mass spectrum of rhP4H. (c) The activity identification mass spectrum of PDI.
