title: Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.
abstract: This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.
authors.affiliation: RIKEN Structural Biology Laboratory, Yokohama, Japan.
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.affiliation: RIKEN Structural Biology Laboratory, Yokohama, Japan. Electronic address: yokoyama@riken.jp.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*; D007553:Isotope Labeling; D008024:Ligands; D008970:Molecular Weight
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands
keywords: Amino acid; Amino acid selective; Cell-free protein synthesis; Escherichia coli cell extract; Mammalian protein; Membrane protein; NMR; Protein complex; Site specific; Stable isotope labeling