title: Improvement of translation efficiency in an Escherichia coli cell-free protein system using cysteine.
abstract: Cell-free protein synthesis systems are powerful tools for protein expression, and allow large amounts of specific proteins to be obtained even if these proteins are detrimental to cell survival. In this report we describe the effect of cysteine on cell-free protein synthesis. The addition of cysteine caused a 2.7-fold increase in the level of synthesized glutathione S-transferase (GST). Moreover, the levels of sulfhydryl group reductants, including reduced glutathione and dithiothreitol (DTT), were increased 1.9- and 1.7-fold, respectively, whereas levels of the disulfide dimers, cystine and oxidized glutathione, were suppressed 87% and 66%, respectively. These trends were also observed for green fluorescent protein (GFP) expression. The addition of cysteine competitively reversed the inhibitory effect of cystine on protein expression. These results suggest that the sulfhydryl group in cysteine plays a crucial role in enhancing protein synthesis, and that the addition of excess cysteine could be a convenient and useful method for improving protein expression.
authors.affiliation: Josai International University, Togane, Chiba 283-8555, Japan. shingaki@jiu.ac.jp
authors.forename: Tomoteru
authors.initials: T
authors.lastname: Shingaki
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nimura
mesh_terms: D002458:Cell Fractionation; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004926:Escherichia coli; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D003553:Cystine; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D019803:Glutathione Disulfide