Rapid screening of E. coli extracts by heteronuclear NMR.
Assessing whether a protein or protein complex is amenable to structural analysis is an important component in the structural genomics effort. In particular, if complete sets of structures for entire genomes are to be obtained within a reasonable time frame, high throughput methodologies for all steps along the way have to be developed. These days, cloning and expression systems are highly optimized and a variety of commercially available vectors can be used. However, heterologous proteins or protein domains expressed in bacteria may not be soluble or correctly folded, necessitating intricate solubilization and refolding schemes prior to structural or functional studies. NMR spectroscopy is an important tool for assessing the solubility, stability, and structural integrity of a gene product. It allows efficient evaluation of many variations of polypeptide length and sequence (without time- and labor-intensive purification/refolding procedures) using 1H-15N-HSQC spectroscopy of samples of 15N-labeled proteins directly from crude E. coli extracts. In addition to screening for particular properties of the expressed protein alone, it is also possible to map intermolecular interactions such as ligand binding. In this unit, the basic methodology for bacterial growth, isotope labeling, and spectroscopic evaluation of the protein structure is provided.
Gronenborn
Angela M
AM
National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland, USA.
D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000295:instrumentation / Q000379:methods; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D006859:Hydrogen