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High‐yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion
Author(s) -
Su PinChuan,
Si William,
Baker Deidre L.,
Berger Bryan W.
Publication year - 2013
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2224
Subject(s) - circular dichroism , membrane protein , fusion protein , bacterial outer membrane , escherichia coli , biophysics , vesicle associated membrane protein 8 , membrane , biochemistry , chemistry , heterologous , heterologous expression , fusion , green fluorescent protein , biology , recombinant dna , gene , linguistics , philosophy
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% α‐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.