title: Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes.
abstract: Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, 305-764, Korea.
authors.forename: Ho-Cheol
authors.initials: HC
authors.lastname: Kim
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, 305-764, Korea.
authors.forename: Kwang-Soo
authors.initials: KS
authors.lastname: Kim
authors.affiliation: Department of Chemical and Biochemical Engineering, Dongguk University, Seoul, Korea.
authors.forename: Taek-Jin
authors.initials: TJ
authors.lastname: Kang
authors.affiliation: Industrial Microbiology & Bioprocess Research Center, Integrated Biorefinery Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeonbuk, Korea.
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.affiliation: Industrial Microbiology & Bioprocess Research Center, Integrated Biorefinery Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeonbuk, Korea.
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology and School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea.
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology and School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea.
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, 305-764, Korea. dmkim@cnu.ac.kr.
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008403:Mass Screening / Q000379:methods; D014176:Protein Biosynthesis; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D012799:Sialyltransferases; D005968:Glutamate Decarboxylase
keywords: cell-free protein synthesis; glutamate decarboxylase; high-throughput expression and screening; pH homeostasis; α-2,3-sialyltransferase