The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.
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 (2.3 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.
Fig. 1. The His8 tag, and the first five fl-cytb5 residues liberated by TEV proteolysis are shown in bold letters, the TEV site is shown in italic letters.
Sobrado P, Goren MA, James D, Amundson CK, Fox BG. "A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles." Protein Expr Purif 58, 229-41 (2008). PubMed ID: 18226920 | Search SBKB Publications portal | PMC Link
Brian G. Fox (firstname.lastname@example.org)
Transmembrane Protein Center
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Last edited:Mon 17 Nov 2014 - 4 years ago