title: A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.
abstract: 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.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Room 141B, 433 Babcock Drive, Madison, WI 53706, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Sobrado
authors.forename: Michael A
authors.initials: MA
authors.lastname: Goren
authors.forename: Declan
authors.initials: D
authors.lastname: James
authors.forename: Carissa K
authors.initials: CK
authors.lastname: Amundson
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D015786:Cytochromes b5 / Q000096:biosynthesis*; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors; D006418:Heme / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D002352:Carrier Proteins; D008081:Liposomes; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D006418:Heme; D015786:Cytochromes b5; D010450:Endopeptidases; C099488:TEV protease