CAJ | 학술논문

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1).方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1 DNA序列,构建表达载体,在大肠杆菌Origami B(DE3)中与硫氧还蛋白Trx A融合表达,并通过盐析和镍柱亲合层析进行纯化.结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28 kDa,占上清总蛋白的50%以上.经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右.结论:复合干扰素在大肠杆菌中的高效可溶表达.
목적:재대장간균중적가용표체화순화인이도소양생장인자1(hIGF-1).방법:근거hIGF-1적안기산서렬화대장간균밀마자편애성,이용중첩연신PCR적방법합성hIGF-1 DNA서렬,구건표체재체,재대장간균Origami B(DE3)중여류양환단백Trx A융합표체,병통과염석화얼주친합층석진행순화.결과:SDS-PAGE분석현시,중조융합단백이가용형식존재,분자량약위28 kDa,점상청총단백적50%이상.경염석화얼주친합층석진행순화,목표단백순도가체도90%좌우.결론:복합간우소재대장간균중적고효가용표체.
Objective:To obtain high level soluble expression and purification of human insulin like growth factor 1 (hIGF - 1) in E.coli.Method: The DNA of hIGF- 1 was amplified by recursive PCR, digested with BamH Ⅰ and Hind Ⅲ, and cloned into pET32a vectors to generate fusions with Trx A.The fusion protein Trx - IGF was expressed in soluble form, and was successfully purifiesd by salt out and Ni-NTA affinity ehromatographye.Result: SDS- PAGE analysis suggested the recombinant fusion protein was expression as soluble form, fusion protein had a molecular weight of 20 kDa, ant its accounted for about 50 % of total protein following cell iysis.The purity of recom-binant protein reached over 90 % through two steps of purification.Conclusion: High level expression of recombinant fusion profion has been achieved in E.coli expression system.