title: Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag.
abstract: Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., 603 WBSB, Baltimore, MD 21205, USA.
authors.forename: Krisna C
authors.initials: KC
authors.lastname: Duong-Ly
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., 603 WBSB, Baltimore, MD 21205, USA. Electronic address: gabelli@jhmi.edu.
authors.forename: Sandra B
authors.initials: SB
authors.lastname: Gabelli
mesh_terms: D000688:Amylose / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013010:Sonication
chemical_list: D002021:Buffers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000688:Amylose; D004229:Dithiothreitol
keywords: Affinity chromatography; Affinity purification; Fast protein liquid chromatography (FPLC); Fusion protein; MBP fusion; Maltose-binding protein (MBP); Recombinant protein