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

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