期刊
出版社
ELSEVIER
DOI: 10.1016/j.jchromb.2013.07.021
关键词
Membrane protein; FhuA; Organic solvent; Extraction; Green solvent
资金
- Excellence Initiative by the German federal and state governments to promote science and research at German universities
- Deutsche Forschungsgemeinschaft (DFG) within the International Research Training Group Selectivity in Chemo- and Biocatalysis - SeleCa
- Deutsche Forschungsgemeinschaft (DFG) [GRK 1166]
beta-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of beta-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; 70 mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay. (c) 2013 Elsevier B.V. All rights reserved.
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