title: 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.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringer Weg 1, Aachen D-52074, Germany.
authors.forename: Stefanie-Joana
authors.initials: SJ
authors.lastname: Tenne
authors.forename: Julia
authors.initials: J
authors.lastname: Kinzel
authors.forename: Marcus
authors.initials: M
authors.lastname: Arlt
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Sibilla
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D003517:Cyclopentanes / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005663:Furans / Q000737:chemistry*; D055772:Green Chemistry Technology / Q000379:methods; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003517:Cyclopentanes; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D005663:Furans; D011994:Recombinant Proteins; D012997:Solvents; C550584:2-methyltetrahydrofuran
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