Cell-free synthesis of 15 N-labeled proteins for NMR studies

Abstract

SummaryModern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 Nlabeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy.IUBMB Life, 57: 615 -622, 2005 Keywords Cell-free protein synthesis; E. coli S30 extract; 15 Nlabeling; NMR spectroscopy; 15 N-HSQC spectrum.

The proofreading exonuclease subunit ε of Escherichia coli DNA polymerase III is tethered to the polymerase subunit α via a flexible linker

“…S30 cell extracts from either E. coli strain Rosetta::λDE3/pRARE (from Novagen, Gibbtown, NJ, USA) or BL21Star::λDE3 (from Invitrogen, Carlsbad, CA, USA) were prepared by the procedure of Pratt (24,30,31), followed by concentration with polyethylene glycol 8000 as described by Kigawa et al (31,32), and were used interchangeably. Cell-free protein synthesis was carried out for 6–7 h either using an autoinduction system that uses plasmid pKO1166 to direct production of T7 RNA polymerase in S30 extracts (33) at 37°C, or a standard coupled transcription/translation system with purified T7 RNA polymerase at 30 or 37°C as described previously (28,31).…”

Cell-free protein synthesis: Applications come of age

“…A key advantage of cell-free systems is that the efficiency of labeled amino acid incorporation, high-protein expression yields, and purity of expressed products in cell-free systems can allow for direct heteronuclear NMR analysis without purification (Morita et al , 2003, Ozawa et al , 2005, Takai et al , 2008). Already, several thousands of protein structures have been determined using cell-free systems (Endo and Sawasaki, 2003).…”

High yield expression of proteins in <i>E. coli</i> for NMR studies

“…those proteins where purification is not feasible like-membrane proteins which pose obstacles to work with [70], viral proteins [69], incorporation of non-natural amino acids [70,71], in studying protein-interactions and also in high throughput proteomics [69,72]. In addition, cell free method provides a means by which target proteins can be decisively screened for NMR studies by means of multiple screening systems [73].…”