Structural genomics methods applied to production of the monotopic membrane protein human cytochrome b5 and in situ delivery to liposomes

Technology

Protein Expression

Summary

Fusion protein vectors developed for high-throughput protein expression as part of the Protein Structure Initiative have been investigated for use in the expression and stabilization of human cytochrome b5, a monotopic membrane protein that must be attached to the cellular membrane for function.

Description

Fusion protein vectors developed for high-throughput protein expression as part of the Protein Structure Initiative have been investigated for use in the expression and stabilization of human cytochrome b5, a monotopic membrane protein that must be attached to the cellular membrane for function. Expression as a fusion to His8-maltose binding protein allowed expression of the full-length cyt b5 (fl-cytb5) as a fully soluble entity. Maintenance of the solubility in E. coli during the time course of expression was associated with high-level incorporation of protoporphyrin IX into the heme domain of the fusion protein. The fl-cytb5 could be liberated from the fusion by site-specific proteolysis, which permitted spontaneous incorporation into membrane vesicles. This work provides a convenient method for the production and high-yield in situ delivery of monotopic membrane proteins to lipid environments.

Figure


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Publication

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 (2007). PubMed ID: 18226920 | Search SBKB Publications portal | PMC Link

Contact

Brian Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Availability

Publication available online.

Link




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Last edited:Wed 16 Jan 2013 - 4 years, 5 months ago