牛肠激酶基因工程菌的构建、高密度发酵、纯化及活性鉴定  

Construction, High-Density Fermentation and Purification of Recombinant Enterokinase Catalytic Subunit in Pichia pastoris

作　　者：李德华[1] 苟兴华 胡海洋 徐琦[2] 刘忠荣 吴洽庆[2] 余晓东[1] 

机构地区：[1]重庆师范大学生命科学院,重庆400047 [2]成都地奥制药集团有限公司,成都610041

出　　处：《应用与环境生物学报》2006年第6期819-823,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家"863"计划项目(2001AA215401)~~

摘　　要：肠激酶(enterokinase,EK)是一种专一识别DDDDK氨基酸序列、并水解K后肽键的丝氨酸蛋白水解酶.根据GenBank序列进行人工合成牛肠激酶轻链基因cDNA,测序正确后将其插入甲醇酵母分泌型载体pPICZαA,得到重组质粒pPICZαA/EKL,重组载体经线性化后转化Pichia pastorisSMD1168H,筛选得到重组牛肠激酶轻链工程菌.分别对重组酵母工程菌进行高密度发酵、rEKL(recombinant enterokinase light chain,rEKL)纯化、N端序列测定、生物学活性鉴定等研究工作.结果表明,发酵上清中rEKL含量高,纯化的rEKL专一性强、无其他杂酶活性、N端15个氨基酸序列与文献报道一致.Enterokinase was a serine protein hydrohyic enzyme that could recognize amino acid sequence DDDDK and cleave the peptide bond after ＂ K＂. eDNA of bovine enterokinase light chain was artificially synthesized based on the sequences in the GeneBank, and inserted into the pPICZαA after validation of obtaining plasmid pPICZαA/EKL, which was transmitted into Pichia pastoris SMD 1168H after linearization, thus the recombinant pichia pastoris strain was obtained by high-density fermentation of recombinant pichia pastoris strain, rEKL （ recombinant enterokinase light chain, rEKL ） purification, N-terminus sequences of EK examination and bioactivity assay. The results demonstrated that rEKL was highly expressed in the culture supernatant with high specificity and the amino acid sequences in N-terminus were consistent with the literature report. Fig 5, Ref 17

关 键 词：肠激酶(enterokinase EK) 甲醇酵母(Pichia pastoris) 高密度发酵 

分 类 号：Q789[生物学—分子生物学]