Cell-free protein production system with the E. coli crude extract for determination of protein folds.
Escherichia coli cell extract-based coupled transcription-translation cell-free system has been developed for large-scale production of protein samples for both X-ray crystallography (selenomethionine substitution) and NMR (stable-isotope labeling). For both cases, higher labeling/substitution efficiency can be achieved compared with the production using cell-based expression system. In addition, as the system is easily adapted to automated and/or high-throughput procedures, it is an especially suitable protein expression method for structural genomics and proteomics project. In this chapter, the procedure for large-scale protein production for structure determination using our E. coli cell-free system is presented.
Kigawa
Takanori
T
RIKEN Systems and Structural Biology Center, Yokohama, Japan.
D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012645:Selenomethionine / Q000378:metabolism; D014158:Transcription, Genetic
D002457:Cell Extracts; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012645:Selenomethionine