title: An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation.
abstract: A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, MS4R0230, Berkeley, CA 94720, USA.
authors.forename: Evan H
authors.initials: EH
authors.lastname: Bursey
authors.forename: Chang-Yub
authors.initials: CY
authors.lastname: Kim
authors.forename: Minmin
authors.initials: M
authors.lastname: Yu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Hung
mesh_terms: D001331:Automation; D002474:Cell-Free System; D003001:Cloning, Molecular; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins