Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1.

ABSTRACT: Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols,

SUBMITTER: Abeyrathne PD 

PROVIDER: S-EPMC5503242 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature