title: FhuA deletion variant Δ1-159 overexpression in inclusion bodies and refolding with Polyethylene-Poly(ethylene glycol) diblock copolymer.
abstract: Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high β-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.
authors.affiliation: Department of Biotechnology (Biology VI), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Dworeck
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Petri
authors.forename: Noor
authors.initials: N
authors.lastname: Muhammad
authors.forename: Marco
authors.initials: M
authors.lastname: Fioroni
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017353:Gene Deletion; D002479:Inclusion Bodies / Q000378:metabolism*; D020959:Polyethylene / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D058849:Protein Refolding; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011092:Polyethylene Glycols; D020959:Polyethylene