Cell-free synthesis of zinc-binding proteins.
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.
Matsuda
Takayoshi
T
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Kigawa
Takanori
T
Koshiba
Seizo
S
Inoue
Makoto
M
Aoki
Masaaki
M
Yamasaki
Kazuhiko
K
Seki
Motoaki
M
Shinozaki
Kazuo
K
Yokoyama
Shigeyuki
S
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*
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