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 ∼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. © 2007 Elsevier Inc. All rights reserved.

Department(s)

Chemistry

Comments

National Institute of General Medical Sciences, Grant U54 GM074901

Keywords and Phrases

Auto-induction; Cytochrome b5; Homo sapiens; Human; Monotopic membrane protein; Peripheral membrane protein; Protein Structure Initiative

International Standard Serial Number (ISSN)

1046-5928

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Apr 2008

PubMed ID

18226920

Link to Full Text

Share

 
COinS