title: Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.
abstract: We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, South Korea.
authors.forename: Yong-Chan
authors.initials: YC
authors.lastname: Kwon
authors.forename: Jae-Kwang
authors.initials: JK
authors.lastname: Song
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D002474:Cell-Free System; D057075:Enzyme Assays / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D014280:Triglycerides; D000637:Transaminases; D008049:Lipase; C431349:lipase B, Candida antarctica; C005830:tributyrin