[High level expression of recombinant human kallistatin in Pichia pastoris and its bioactivity].
In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Huang
Xiaoping
X
Institute of Molecular Medicine, Huaqiao University, Quanzhou, China.
Wang
Xiao
X
Dong
Hao
H
Zhao
Xiaofeng
X
Li
Zhaofa
Z
Wang
Qizhao
Q
Xu
Ruian
R
Diao
Yong
Y
D000975:Antioxidants / Q000494:pharmacology; D018076:DNA, Complementary; D018274:Electroporation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics
D000975:Antioxidants; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015843:Serpins; C051324:kallistatin