Research Article: Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1

Date Published: July 10, 2017

Publisher: Public Library of Science

Author(s): Priyanka D. Abeyrathne, Nikolaus Grigorieff, Dimitrios Fotiadis.


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, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.

Partial Text

X-ray crystallography, nuclear magnetic resonance (NMR), electron crystallography, and single-particle electron cryo-microscopy (cryo-EM) have all been used to obtain high-resolution structural information from both soluble and membrane proteins. However, using these techniques to resolve the structures of membrane proteins is especially challenging. The purification process, necessary to prepare suitable samples for high-resolution structural imaging, is particularly labor-intensive for membrane proteins, due to their hydrophobicity and reduced stability in solution. In response to these challenges, work in the field has moved towards leveraging fluorescence size-exclusion chromatography (FSEC) to screen detergents and buffer conditions in a high-throughput manner [1–3]. However, producing sufficient quantities of stable protein with minimal contaminants continues to be a major bottleneck [4].

The general approach to preparing membrane protein for high-resolution structural studies involves: (1) cloning and expression; (2) solubilization; (3) purification; (4) detergent exchange; and (5) assessment of stability, quality, and purity. We systematically optimized each of these preparation steps.

We conducted a systematic examination of several variables involved in the preparation of membrane protein samples for high-resolution structural studies including expression system, solubilization, purification and detergent exchange protocols, and assays for stability and impurity. We found that the detergent types was a key variable, as some detergents can contribute to contaminant co-purification [4]. Combining these considerations with techniques such as SEC and affinity resins can reduce the presence of contaminants. However, it often remains difficult to detect contaminant proteins before they are analyzed further for structural studies. When working with small membrane proteins, such as ClC-rm1, it is particularly important to remove all contaminants as even small molecules can interefere with cryo-EM structural studies. In these studies, we found that the small contaminant protein AcrB resisted purifictation by all standard methods. Thus, we turned instead to an expression system using the E. coli strain lacking AcrB (JW0451-2, ΔacrB). Additionally, we incorporated the eGFP to the C-terminus of ClC-rm1 as it promotes proper folding during expression of membrane proteins and increases the solubility during solubilization of membrane proteins. These strategies allowed us to express and purify properly folded, contaminant-free homogeneous ClC-rm1 protein suitable for high-resolution data collection. The presence of the eGFP tag will also help with the initial particle alignment as it adds clearly identifiable features to distinguish the cytoplasmic domian from the trnasmembrane domain of the protein. The tag may interfere with the alignment at high resolution due to its flexibility and density masking may have to be used to obtain accurate alignments. Our present study can serve as a guide when establishing new purification protocols, particularly of small membrane proteins, for cryo-EM and other quantitatiove structural studies.