title: Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems.
abstract: Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.
authors.affiliation: Harvard Institute of Proteomics, 320 Charles street, Cambridge, MA 02141, USA.
authors.forename: T V S
authors.initials: TV
authors.lastname: Murthy
authors.forename: Weilin
authors.initials: W
authors.lastname: Wu
authors.forename: Q Q
authors.initials: QQ
authors.lastname: Qiu
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Shi
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
authors.forename: Leonardo
authors.initials: L
authors.lastname: Brizuela
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002474:Cell-Free System / Q000737:chemistry*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins