title: T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination.
abstract: T4-lysozyme (T4L) fusion was introduced in the intracellular loop of a G protein-coupled receptor (GPCR) of human formyl peptide receptor 3 (FPR3), and the ability of T4L fusion to be used in the production of human FPR3 for structural determination was evaluated in this work. The T4L variant of human FPR3 termed FPR3-T4L was expressed in stable tetracycline-inducible HEK293 cells. A systematic detergent screening showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify FPR3-T4L from HEK293 cells. Immunoaffinity purification in combination with gel filtration was employed to purify the T4L-fused receptor to high homogeneity. The final yield of the human FPR3-T4L monomer from 2 g of cells was 0.2 mg. Circular dichroism spectroscopy indicated that the receptor adopted a correct secondary structure after purification, while ligand binding measurement indicated that the receptor was functional. Thus, the presence of T4L fusion did not evidently disturb the expression in HEK293 cells, proper folding, and functionality of human FPR3. Our study of evaluating T4L fusion for the recombinant production of human formyl peptide receptor would facilitate ongoing efforts in the structural characterization of GPCRs.
authors.affiliation: State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao, 266580, People's Republic of China, wangxq001@upc.edu.cn.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Cui
authors.forename: Jiqian
authors.initials: J
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000737:chemistry; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D044042:Receptors, Formyl Peptide / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003902:Detergents; C469985:FPR3 protein, human; D044042:Receptors, Formyl Peptide; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010767:Phosphorylcholine; C028810:dodecylphosphocholine; D009113:Muramidase