2-Methyltetrahydrofuran and cyclopentylmethylether: two green solvents for efficient purification of membrane proteins like FhuA

Abstract

β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.

Keywords: 2-MeTHF; 2-methyltretahydrofuran; 3,3′,5,5′-tetramethylbenzidine; BCA-assay; CD; CM; CPME; Extraction; FhuA; Green solvent; HRP; IPTG; Membrane protein; Organic solvent; PE-PEG; STD; TMB; TonB; bicinchoninic acid assay; chloroform/methanol; circular dichroism; cyclopentylmethylether; ferric hydroxamate uptake protein component A; horse radish peroxidase; isopropyl-β-d-thiogalactopyranoside; n-octyl-poly-oxyethylene; oPOE; polyethylene-polyethyleneglycol; protein to transduce cytoplasmic energy to the outer membrane; standard extraction.