TAILIEUCHUNG - Báo cáo khoa học: 15 N-Labelled proteins by cell-free protein synthesis Strategies for high-throughput NMR studies of proteins and protein–ligand complexes

[ 15 N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [ 15 N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [ 15 N]-HSQC spectra by amino acid type selective [ 15 N]-labelling. | ễFEBS Journal MINIREVIEW 15N-Labelled proteins by cell-free protein synthesis Strategies for high-throughput NMR studies of proteins and protein-ligand complexes Kiyoshi Ozawa Peter S. C. Wu Nicholas E. Dixon and Gottfried Otting Research Schoolof Chemistry Australian National university Canberra ACT Australia Keywords cell-free protein synthesis combinatorial labelling 15N-HSQC 15N-labelled amino acids protein-ligand interactions Correspondence G. Otting Research Schoolof Chemistry Australian National university Canberra ACT Australia Fax 61 261250750 Tel 61 261256507 E-mail Website http go Received 9 May 2006 accepted 23 June 2006 doi 15N -heteronuclear single quantum coherence HSQC spectra provide a readily accessible fingerprint of 15N -labelled proteins where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of 15N -HSQC spectra by amino acid type selective 15N -labelling. The samples can be measured without chromatographic protein purification dilution of isotopes by transaminase activities are suppressed and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all 15N -HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking 15N -HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h including protein production and NMR measurement. Introduction Cell-free protein synthesis in both the Escherichia coli coupled transcription-translation system and the wheat germ translation system has been remarkably improved so that milligram quantities

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