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High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion.

ABSTRACT: Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full-length RAMP1 is composed of approximately 90% ?-helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20-60°C). Thus, our approach provides a useful, complementary approach to achieve high-yield, full-length membrane protein overexpression for biophysical studies.

SUBMITTER: Su PC

PROVIDER: S-EPMC3610049 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion.

Su Pin-Chuan PC Si William W Baker Deidre L DL Berger Bryan W BW

Protein science : a publication of the Protein Society 20130221 4

Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to h ...[more]

PMID: 23345122

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