Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

重组a-氨基酸酯水解酶合成头孢曲嗪

为了探索酶法合成头孢曲嗪的产业化工艺路线,从红纹黄单胞菌Xanthomonas rubrillineans中克隆-氨基酸酯水解酶基因全序列,转化入大肠杆菌中表达。以头孢曲嗪的合成转化率为指标,分别考察纯化的重组-氨基酸酯水解酶合成头孢曲嗪的最适温度、最适pH和最佳底物摩尔比。经聚丙烯酰胺凝胶电泳分析,重组-氨基酸酯水解酶的单体分子量为70 kDa。催化合成头孢曲嗪的最适pH为(6.0±0.1),最适温度为36℃。底物浓度约为7-ATTC 30 mmol/L、HPGM HCl 120 mmol/L,酶用量22 U/mL时,头孢曲嗪的转化率达到64.3%。结果为优化酶法合成头孢曲嗪的产业化工艺奠定了基础。     

Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst

Bioprocess and Biosystems Engineering - A method for the synthesis of β-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of...