Production of human intestinal trefoil factor in Pichia pastoris  

作　　者：孙勇 彭曦 吕尚军 张勇 汪仕良 

机构地区：[1]State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burns,Southwest Hospital,Third Military Medical University,Chongqing 400038,China

出　　处：《Journal of Medical Colleges of PLA(China)》2006年第4期203-208,共6页中国人民解放军军医大学学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China (No. 30200294)

摘　　要：Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.Objective:To construct a Pichia pastoris (P. pastoris) expression vector of human intestinal trefoil factor (hITF) and study its expression and purification procedures. Methods:hITF gene encoding mature peptide was modified with a polyhistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZαA at the downstream of theα-mating factor signal. After gene sequencing, the recombinant pGAPZαA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride. rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGE and Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results:The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing. rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion: hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.

关 键 词：intestinal trefoil factor Pichia pastoris secretory expression PURIFICATION 

分 类 号：R285[医药卫生—中药学]