title: Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.
abstract: As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Natsuko
authors.initials: N
authors.lastname: Matsuda
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Rie
authors.initials: R
authors.lastname: Nakajima
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanaka
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D023281:Genomics; D009682:Magnetic Resonance Spectroscopy; D016133:Polymerase Chain Reaction; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases