摘要
用RT＿PCR扩增肠激酶轻链(EKLC,EnterokinaseLightChain)的cDNA,并克隆至酵母分泌型表达质粒pPICZαA中.将重组质粒pPICZαA/EKLC转化至表达宿主pichiapastorisGS115,经甲醇诱导,目标蛋白EKLC表达并分泌至酵母培养基中.采用Zn＿Sepharose亲和层析一步纯化,从每升培养液可获得1.6mgEKLC,其纯度达90%以上.酶反应动力学结果表明,EKLC对小分子底物Gly＿Asp＿Asp＿Asp＿Asp＿Lys＿β＿naphthylamide的Km为(753±42)mol/L,kcat为(31.5±3.8)s-1.对硫氧化还原蛋白-脑钠素融合蛋白(Trx＿hBNP)的酶解作用显示,当酶与底物重量比大于1∶100时,经37℃保温5h后,超过75%的融合蛋白被裂解.上述结果表明,EKLC能够特异识别并水解Asp＿Asp＿Asp＿Asp＿Lys＿肽键,是一种能将融合蛋白中目标蛋白与载体蛋白裂解释放的有效工具酶.
Enterokinase (EK) is a protease at the intestinal brush border that catalyzes the conversion of trypsinogen into active trypsin. This conversion initiates the activation of other pancreatic proteolytic zymogens such as chymotrypsinogen, procarboxypeptidase and proelastase. Enterokinase deficiency seriously impairs protein absorption and leads to severe malabsorption, diarrhoea, and vomiting. Bovine enterokinase is a disulfide linked heterodimer, composed of an 115 kD heavy chain and a 35 kD light chain. The heavy chain contains an amino terminal membrane spanning segment which is involved in substrate recognition and inhibitor specificity. The light chain containing the serine protease domain carries the full catalytic activity. EK is a site specific protease that cleaves after lysine at the recognition site Asp Asp Asp Asp Lys. It has been widely used to release recombinant polypeptides from fusion proteins.We describe here the cloning and expression of the bovine enterokinase light chain (EKLC) in the yeast Pichia pastoris. The cDNA encoding EKLC was amplified by RT PCR and inserted into yeast secretory expression plasmid pPICZαA. The plasmid was then transformed into host cells Pichia pastoris GS 115, and after induced with methonal, EKLC was expressed and secreted into the culture medium. 1.6 mg EKLC with a homogeneity greater than 90% was obtained after only a single step purification with Zn Sepharose affinity column. Kinetic analysis showed that the hydrolysis of Gly Asp Asp Asp Asp Lys β naphthylamide was catalyzed by EKLC with Km of (753±42) μmol/L and kcat of (31.5±3.8) s^(-1). The cleavage efficiency and specificity of EKLC for the sequence Asp Asp Asp Asp Lys were further tested by digesting thioredoxin natriuretic peptide (Trx BNP) fusion protein. It was found that more than 75 % of the fusion protein was cleaved after incubation at 37 ℃ for 5 hours with EKLC at a ratio of 100:1 (w/w). These results indicate that EKLC is a valuable tool enzyme which can be used to effectively release the target polypeptide from a fusion protein.
出处
《南京大学学报（自然科学版）》
CAS
CSCD
北大核心
2004年第1期41-48,共8页
Journal of Nanjing University（Natural Science）
基金
国家重大科技专项"创新药物和中药现代化"(2002AA2Z3451)
