title: Cell-free synthesis of zinc-binding proteins.
abstract: Cell-free protein synthesis has become one of the standard methods for protein expression. The cell-free method is suitable for the synthesis of a protein that requires a ligand for its enzymatic activity and/or structure formation and stabilization, since it is an open system, which allows us to add the proper ligand to the reaction mixture. A large number of proteins that require zinc for their function are involved in diverse cellular processes, including transcription, DNA replication, metabolism, and cell signaling. In this study, we analyzed the effects of zinc on the cell-free synthesis of plant-specific zinc-binding transcription factors. The solubility and/or stability of the proteins were significantly increased in the presence of the proper concentration of zinc during the cell-free reaction. NMR analyses confirmed that correctly folded proteins were synthesized by the cell-free method. These results indicate that the cell-free method can be used to synthesize correctly folded and functional zinc-binding proteins.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Seizo
authors.initials: S
authors.lastname: Koshiba
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Masaaki
authors.initials: M
authors.lastname: Aoki
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Motoaki
authors.initials: M
authors.lastname: Seki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D023281:Genomics / Q000379:methods*; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
chemical_list: D029681:Arabidopsis Proteins; D009587:Nitrogen Isotopes; D009687:Nuclear Proteins; D010940:Plant Proteins; D012097:Repressor Proteins; C416221:SPL protein, Arabidopsis; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D015032:Zinc