摘要
目的对链霉亲和素(SA)进行定点突变,降低与生物素之间的亲和力,制备SA突变体琼脂糖凝胶柱,并摸索最适洗脱条件,以达到纯化生物素修饰蛋白的目的。方法原核表达SA突变体蛋白使用Ni-NTA纯化树脂进行纯化,将SA突变体蛋白与溴化氰活化琼脂糖凝胶4FF(CNBr-activated Bestarose TM 4FF)偶联制备凝胶柱。生物素修饰蛋白与凝胶柱孵育后,以不同浓度NaOH对生物素修饰蛋白进行洗脱回收,ELISA检测SA突变体蛋白亲和力。通过Western blot法、考马斯亮蓝染色检测纯化效果。结果成功表达并纯化五种SA突变体,并制备凝胶柱。ELISA结果计算得到SA突变体对生物素修饰蛋白的亲和常数相较野生型SA均有所降低。将生物素修饰蛋白与五种凝胶柱相结合,通过Western blot法和考马斯亮蓝染色结果筛选出SA突变体M2凝胶柱可以在10 mmol/L NaOH洗脱条件下,纯化出生物素修饰蛋白,并且SA突变体M2凝胶柱能够对生物素修饰蛋白进行再次纯化。结论成功制备了SA突变体琼脂糖凝胶柱,为生物素修饰蛋白的纯化提供了可行性方案。
Objective To make a site-directed mutation of streptavidin(SA)to reduce its affinity to biotin,to prepare a streptavidin mutant agarose gel column,and to find the optimal elution conditions to purify the biotin-modified protein.Methods The streptavidin mutant protein expressed in prokaryotes was purified using Ni-NTA resin,and the purified SA mutant protein was coupled with CNBr-activated BestaroseTM 4FF agarose gel to prepare the column.The biotin-modified protein was incubated with the gel column,and was eluted and recovered using NaOH at various concentrations.The purity of protein was detected by Western blotting and Coomassie brilliant blue staining.Results Five SA mutants were expressed and purified,and gel columns were successfully prepared.The results of ELISA showed that the affinity of SA-mutant to biotin-modified protein was lower than that of wild type SA.By purifying the biotin-modified protein with different gel columns,we screened out the SA mutant M2 gel column that could purify the biotin-modified protein with 10 mmol/L NaOH.And the M2 gel column could be used to re-purify biotin-modified protein.Conclusion The SA mutant agarose gel column has been successfully prepared,which provides a feasible and reliable option for the purification of biotin-modified protein.
作者
陈园坤
伦新新
李昂
王鹏举
张仪婷
赵晶
王辉
阎博
杨安钢
CHEN Yuankun;LUN Xinxin;LI Ang;WANG Pengju;ZHANG Yiting;ZHAO Jing;WANG Hui;YAN Bo;YANG Angang(School of Laboratory Medicine,Xinxiang Medical University,Xinxiang 453003;Department of Biochemistry and Molecular Biology,Air Force Medical University,Xi'an 710032,China;State Key Laboratory of Cancer Biology,Department of Immunology,Air Force Medical University,Xi'an 710032,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2020年第4期304-309,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81630069,81972870)。
