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A suitable and effective stepwise oxidative refolding procedure for highly-cationic tetrameric avidin in nucleic acid free conditions.
Biotechnology Progress ( IF 2.5 ) Pub Date : 2020-05-28 , DOI: 10.1002/btpr.3031
Shuichiro Kimura 1 , Koreyoshi Imamura 1 , Junichiro Futami 2
Affiliation  

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S‐cationization procedure was successfully employed for large‐scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

中文翻译:

在无核酸条件下用于高阳离子四聚体亲和素的合适且有效的逐步氧化重折叠程序。

需要优化条件以将重组蛋白从细菌包涵体重新折叠为其生物活性构象。在这项研究中,我们发现了阳离子四聚体鸡抗生物素蛋白有效重折叠的两个关键要求。第一步是从细菌包涵体中去除核酸污染物。剩余核酸和蛋白质之间的静电相互作用在重折叠过程中强烈增强了蛋白质的聚集。半胱氨酸特异性可逆S阳离子化程序已成功用于大规模制备无核酸变性蛋白质,无需纯化标签系统。第二步是在透析去除变性剂中重折叠之前形成分子内二硫化物。二硫化物完整单体抗生物素蛋白显示在重折叠过程中有效形成生物活性四聚体构象。使用这种优化的重折叠程序,成功制备了被设计为生物素化蛋白的细胞内递送载体的高阳离子亲和素衍生物。
更新日期:2020-05-28
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