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Expressioneering™ Technology Accelerates Functional Expression and Crystallization of GPCRs for Drug Discovery
Author(s) -
Sen Saurabh,
Franz Laura,
Mead David,
Steinmetz Eric
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1001.5
Subject(s) - g protein coupled receptor , computational biology , cloning (programming) , transmembrane protein , drug discovery , transmembrane domain , lipid bilayer fusion , biology , fusion protein , microbiology and biotechnology , receptor , bioinformatics , signal transduction , membrane , computer science , biochemistry , gene , recombinant dna , programming language
Structural biology of membrane proteins and GPCRs has seen major advances over the past few years, but overcoming the challenges of functional GPCR expression and structural stabilization are still time‐consuming and expensive. Our approach combines a powerful cloning strategy with novel transmembrane guides and co‐expressed helper proteins to significantly increase functional receptor expression and membrane insertion in E. coli . Lucigen's Expressioneering™ technology is a rapid and simple recombination based expression cloning method. We present here the use of this technology to optimize GPCR expression through fusion to a variety of transmembrane guides and novel visualization tags. We have been successful in obtaining conformationally active functional GPCRs with this system. We will also discuss unique new strategies for conformational stabilization of GPCRs through use of novel fusion partners and interacting proteins. Preliminary data using a novel fluorescent protein in the IL3 demonstrates retention of functional activity and may promote GPCR crystallization. Data obtained using Lucigen's Expresso CMV system are comparable with those obtained from the bacterial system.