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

作者：Sobrado Pablo; Goren Michael A; James Declan; Amundson Carissa K; Fox Brian G^*

来源：Protein Expression and Purification, 2008, 58(2): 229-241.

DOI：10.1016/j.pep.2007.11.018

• protein structure initiative
• Homo sapiens
• human
• cytochrome b5
• peripheral membrane protein
• monotopic membrane protein
• auto-induction

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 similar to 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg 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.

• 出版日期2008-4