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Protein Expr Purif. 2008 Apr;58(2):229-41. doi: 10.1016/j.pep.2007.11.018. Epub 2007 Dec 15.

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.

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Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Room 141B, 433 Babcock Drive, Madison, WI 53706, USA.


A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

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