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UBC Theses and Dissertations

A strategy to make soluble integral membrane proteins in vivo Won, Han-Sol

Abstract

Membrane proteins play significant roles in fundamental biological processes, such as transport of molecules across the membrane, triggering intracellular signaling, maintenance of cell structure and utilization of energies. They are known to comprise about 30 % of genes of entire genome; they constitute around 60 % of current drug targets. Despite their importance, our knowledge about membrane proteins lags far behind that about the soluble proteins. This is chiefly because of their nature that hydrophobic domains of integral membrane proteins are embedded within the phospholipid bilayer and because of the fact that they are generally unstable following extraction from their native membrane environment by detergents. It is also true that available techniques for purifying, analyzing and handling membrane proteins are optimized for water-soluble proteins. Hence, a strategy to solubilize membrane proteins in vivo in structurally relevant conformations by fusing membrane proteins with membrane scaffold protein (MSP) could be an alternative to detergent extraction and in vitro solubilization, allowing the direct expression of soluble membrane proteins in living cells. The MSP, which has an amphipathic helical domain, is thought to protect or shield the hydrophobic transmembrane domain of membrane proteins by sequestering them from aqueous environment. To explore this strategy, an ATP binding cassette (ABC) transporter, MsbA, a known lipid flippase was initially chosen as a model protein. MsbA was fused to maltose binding protein (MBP) and MSP at its N-terminus to increase its expression level and to promote solubilization respectively. It was shown that MsbA fusion construct (MBP-MSP-MsbA) was produced in an appreciable amount in water soluble form post detergent wash in the oligomerization state of a functional dimer and was able to hydrolyze ATP.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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