嵌有非天然氨基酸蛋白质的生物合成及其纯化鉴定

Biosynthesis,Purification and Identification of Protein Embedded with Unnatural Amino Acid

基于基因密码子扩展技术,构建能够嵌入非天然氨基酸pBpa蛋白质的大肠杆菌BL21(DE3)双质粒原核表达工程菌株,原核表达后,通过Ni-NTA纯化介质的初步纯化以及AKTA蛋白纯化系统的Superdex75分子筛进一步精细纯化,获得了在IL-1β的5号预设位点上嵌入了非天然氨基酸pBpa的嵌合体蛋白,并且通过质谱确认pBpa在预设位点的精准插入.本研究证明基于密码子扩展技术构建的双质粒工程菌可以将非天然氨基酸pBpa插入到IL-1β的特定位点,能够生物合成并纯化出大量的非天然氨基酸嵌合体蛋白质,这为其他非天然氨基酸或其他功能蛋白及其特异性位点的非天然氨基酸嵌合蛋白的生物合成奠定了基础.

In this study,based on codon expansion technology,we constructed E.coli BL21(DE3)double plasmid prokaryotic expression engineering strain,which can embed protein with the unnatural amino acid pBpa.After prokaryotic expression,the unnatural amino acid pBpa protein was obtained by preliminary purification of Ni-NTA purification medium and further fine purification of Superdex75 molecular sieve of AKTA protein purification system.The chimeric protein of pBpa was identified by mass spectrometry.This study demonstrated that the two plasmid engineering bacteria constructed based on codon extension technology can insert the unnatural amino acid pBpa into the specific site of IL-1β,and can biosynthesize and purify a large number of unnatural amino acid chimeric proteins,which laid a foundation for the biosynthesis of other unnatural amino acids or other functional proteins and their specific site unnatural amino acid chimeric proteins.