title: Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor.
abstract: G-protein coupled receptors (GPCRs) are inherently dynamic membrane protein modulators of various important cellular signaling cascades. The apelin receptor (AR or APJ) is a class A GPCR involved in numerous physiological processes, implicated in angiogenesis during tumour formation and as a CD4 co-receptor for entry of human immunodeficiency virus type 1 (HIV-1) to cells. Due to the lack of efficient methods to produce full-length GPCRs enriched with nuclear magnetic resonance (NMR) active (15)N, (13)C, and (or) (2)H isotopes, small GPCR fragments typically comprising 1-2 transmembrane segments are frequently studied using NMR spectroscopy. Here, we report successful overexpression of transmembrane segments 1-3 of AR (AR_TM1-3) in the C41(DE3) strain of Escherichia coli using an AT-rich gene tag previously reported to enhance cell-free expression yields. The resulting protein, with 6 additional N-terminal residues due to the expression tag, was purified using high-performance liquid chromatography (HPLC). Far UV circular dichroism spectropolarimetry demonstrates that AR_TM1-3 has the predicted ~40% α-helical character in membrane-mimetic environments. (1)H-(15)N HSQC NMR experiments imply amenability to high-resolution NMR structural characterization and stability in solution for weeks. Notably, this small expression tag approach may also be generally applicable to other membrane proteins that are difficult to express in E. coli.
authors.affiliation: a Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
authors.forename: Aditya
authors.initials: A
authors.lastname: Pandey
authors.forename: Muzaddid
authors.initials: M
authors.lastname: Sarker
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
authors.forename: Jan K
authors.initials: JK
authors.lastname: Rainey
mesh_terms: D000075244:Apelin Receptors; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: C104575:APLNR protein, human; D000075244:Apelin Receptors; D003062:Codon; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins
keywords: CD spectroscopy; GPCR; NMR spectroscopy; RCPG; apelin receptor; expression et purification de protéines membranaires; membrane protein expression and purification; récepteur de l’apeline; spectroscopie RMN; spectroscopie par DC