摘要
目的 :提高Klenow酶的热稳定性和储藏稳定性。方法 :利用PCR的方法扩增DH5α染色体DNA ,构建编码Klenow和人血清白蛋白第三功能区 (HSA D3)的融合蛋白表达质粒 ,并在大肠杆菌中获得高效表达。分别对Klenow酶和Klenow HSA融合蛋白进行纯化 ,并进行活性及稳定性的研究。结果 :发现Klenow HSA融合蛋白在大肠杆菌中呈可溶性表达。体外实验证实这种融合蛋白具有与Klenow聚合酶相同的活性 ,其稳定性明显提高。结论 :融合蛋白确实能增加Klenow酶的热稳定性和储存稳定性 。
Objective: To enhance Klenow's thermostability and storage stability. Methods: Two expression plasmids have been constructed: One encoding large fragment of DNA polymerase Ⅰ (Klenow) and the other the fusion protein consisting of Klenow fragment and domain 3 of human serum albumin named as pKpL Klenow HSA. Two sorts of protein were purified. Their activity and stability were studied. Results: It was discovered that this soluble fusion protein had expressed in E.coli . This fusion protein had polymerase activity like Klenow. Klenow HSA was much more stable than Klenow at 37 ℃. Conclusion: The fusion protein does increase Klenow's thermostability and prolong its storage stability. It's provided basis for novel enzyme research.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2002年第6期684-687,共4页
Journal of Peking University:Health Sciences
