论文部分内容阅读
Membrane proteins (MPs) are key targets for pharmacological intervention because of their vital functions.Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins.We developed a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner.A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products.The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption.We applied this approach to produce a functional integral membrane protein, undecaprenyl pyrophosphate phosphatase (UppP) and studied this MP by extensive mutagenesis and biochemical approaches.A plausible structural model was proposed.In addition, the crystal structures of another BR were solved at high resolution which allowed us to explain the activity under acidic condition.Those results will be presented.