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.

journal_name

Protein Expr Purif

authors

Sobrado P,Goren MA,James D,Amundson CK,Fox BG

doi

10.1016/j.pep.2007.11.018

subject

Has Abstract

pub_date

2008-04-01 00:00:00

pages

229-41

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(07)00296-3

journal_volume

58

pub_type

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