title: A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.
abstract: Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32310, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Hu
authors.forename: Huajun
authors.initials: H
authors.lastname: Qin
authors.forename: Fei Philip
authors.initials: FP
authors.lastname: Gao
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Cross
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification*; D015854:Up-Regulation
chemical_list: D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins