Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

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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.


2-mEt — 2-mercaptoethanol; AcCoA — acetyl coenzyme A; BL21 CP — BL21 codon-plus RIL; CAT — chloramphenicol acetyl transferase; CK — creatine kinase; Cm — chloramphenicol; DEPC — diethylpyrocarbonate; CP — creatine phosphate; DTNB — 5,5′-dithiobis-2-nitrobenzoic acid; DTT — dithiothreitol; Folinic acid — L(−)-5-formyl-5,6,7,8-tetrahydrofolic acid; KGlu — polyethyleneglycol; PEG — potassium glutamate; PEP — phospho-enolpyruvate; PK — pyruvate kinase.