Formate-nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives

摘要

The formate-nitrite transporter family is composed of integral membrane proteins that possess six to eight α-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to >95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly α-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer.