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 E. coli when using MBP. In addition, the proteolysis of the fusions were performed on the membrane without solublilization 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.

Expression and purification of the membrane enzyme selenoprotein K

“…Expression level and solubility of membrane proteins are enhanced by the use of fusion proteins that can be cleaved after production [28, 29]. The selected affinity tags: a hexahistidines, SUMO, pMBP, and cMBP tags, were previously reported to be successful for the expression of membrane proteins or difficult targets [30, 31]. Since SelK’s active site is located at the C-terminal, we constructed N-terminal fusions that could be separated from SelK following expression using TEV protease (see bacterial expression vectors in Fig.…”

“…In each case, the Sec was mutated to a Cys (U92C) to assist the protein production. A Sec to Cys substitution is necessary to circumvent the unique requirements for Sec incorporation in proteins (it shares the TGA codon with a stop signal [30]). Since sulfur and selenium share many physicochemical properties [32], the substitution does not otherwise interfere with structural integrity, oligomerization state, or protein stability [23].…”

“…The best expression was observed in the case of the cMBP fusion (also called the mature MBP), a 42 kDa protein that increases solubility and acts as a chaperone assisting protein folding. cMBP-fusion has been reported to be a particularly successful strategy for increasing expression of membrane proteins [30]. The remainder of this manuscript describes optimization of expression and purification using the cMBP-SelK fusion.…”

“…Usually purification strategies for cMBP fused to transmembrane proteins do not utilize detergents during extraction from cells, since the fusion protein is typically soluble [30]. MBP-SelK is soluble and localized to the cytoplasm of E. coli , but following extraction, it aggregated slowly during purification in the absence of detergents.…”

Membrane protein structure from rotational diffusion

“…A very common strategy to express membrane proteins with between one and seven trans-membrane helices in E. coli is the use of fusion proteins. Functional membrane proteins have been successfully expressed using the N-terminal fusion to the E. coli maltose-binding protein (MBP) that targets the protein to the periplasmic membrane [70,74]. The disadvantage of this approach is that toxic effects associated with membrane insertion of large amounts of heterologously overexpressed proteins often limit cell growth.…”