title: High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system.
abstract: The overexpression of subunit b of F(1)F(0) adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 microg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F(1)F(0) ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.
authors.affiliation: Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Jiazhang
authors.initials: J
authors.lastname: Lian
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.forename: Jin
authors.initials: J
authors.lastname: Cai
authors.forename: Ming
authors.initials: M
authors.lastname: Wu
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D002474:Cell-Free System; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D010957:Plasmids; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000235:genetics; D012995:Solubility
chemical_list: D003902:Detergents; D021122:Protein Subunits; D025243:Bacterial Proton-Translocating ATPases