Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.
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.
Kigawa
Takanori
T
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Yabuki
Takashi
T
Matsuda
Natsuko
N
Matsuda
Takayoshi
T
Nakajima
Rie
R
Tanaka
Akiko
A
Yokoyama
Shigeyuki
S
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
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases