tailieunhanh - Báo cáo khoa học: Optimization of an Escherichia coli system for cell-free synthesis of selectively 15N-labelled proteins for rapid analysis by NMR spectroscopy

Cell-free protein synthesis offers rapidaccess toproteins that are selectively labelledwith [ 15 N]aminoacids andsuitable for analysis by NMR spectroscopy without chromatographic purification. A system based on anEscherichia colicell ex-tractwas optimizedwith regard toproteinyieldandminimal usage of 15 N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to mg of human cyclophilin A per mL of reactionmedium were obtained by expression of a synthetic gene. . | Eur. J. Biochem. 271 4084-4093 2004 FEBS 2004 doi Optimization of an Escherichia colisystem for cell-free synthesis of selectively 15N-labelled proteins for rapid analysis by NMR spectroscopy Kiyoshi Ozawa Madeleine J. Headlam Patrick M. Schaeffer Blair R. Henderson Nicholas E. Dixon and Gottfried Otting Research School of Chemistry Australian National University Canberra Australia Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with 15N amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage k pR and pL promoters when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples each selectively labelled with a different 15N-enriched amino acid were produced and analysed directly by NMR spectroscopy after ultracentri fugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for 15N Asp where an enzyme in the cell extract efficiently converted Asp fi Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system. Keywords cell-free protein .