Metalloproteins: structure and function-A set of Escherichia coli amino acid auxotrophic expression host strains for selectively labelled metalloenzyme research

摘要

Specific contributions of particular residues in the reaction mechanisms and/or folding dynamics of a metalloenzyme of interest can be best addressed by using magnetic resonance (e.g. NMR and EPR) and vibrational (e.g. FTIR and resonance Raman) spectroscopic techniques, often aided by X-ray crystallographic analysis. In this work, we report a set of Escherichia coli C43(DE3) and/or BL21(DE3) based amino acid auxotrophic expression host strains suitable for these studies. Currently, our E. coli expression host strain collection may be used for selective ~(15)N and/or ~(13)C labelling of 17 different amino acids, except for serine, aspartate and glutamate. These strains may be applied for producing recombinant archaeal and bacterial metalloproteins when a plasmid carrying extra copies of tRNA genes for E. coli rare codons is incorporated. Examples of our successful application of some of these strains in 2D pulsed EPR (HYSCORE) analysis of ~(15)N or ~(13)C amino acid-labelled iron-sulfur proteins will be presented in the poster.