Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter.
We have modified the cell-free coupled transcription/translation system of bacteria. The cell-free extract of Pseudomonas fluorescens was used for translation instead of Escherichia coli. In addition, transcription of the target gene was regulated by CspA promoter with endogenous RNA polymerase instead of by T7 promoter with exogenous T7 RNA polymerase. We could increase the yields of soluble proteins using different combinations of the S30 extract and the promoter and different temperatures for protein synthesis. Increasing the variety of synthesis systems allows production of large quantities of soluble proteins. In order to carry out efficient cell-free protein synthesis, versatile pCop-plasmids carrying CspA promoter were constructed and these plasmids were applicable to expression of recombinant proteins in E. coli cells.
Nakashima
Nobutaka
N
Proteolysis and Protein Turnover Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Sapporo 062-8517, Japan. n-nakashima@aist.go.jp
Tamura
Tomohiro
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011551:Pseudomonas fluorescens / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins