title: Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.
abstract: GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding.
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Tai-Yun
authors.initials: TY
authors.lastname: Yang
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Nien-Yi
authors.initials: NY
authors.lastname: Chiang
authors.affiliation: Division of Rheumatology, Allergy and Immunology, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan.
authors.forename: Wen-Yi
authors.initials: WY
authors.lastname: Tseng
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Hsiao-Lin
authors.initials: HL
authors.lastname: Pan
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Yen-Ming
authors.initials: YM
authors.lastname: Peng
authors.affiliation: School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital at Lin-Kuo, Kwei-Shan, Tao-Yuan, Taiwan.
authors.forename: Jiann-Jong
authors.initials: JJ
authors.lastname: Shen
authors.affiliation: Department of Internal Medicine, Taoyuan Armed Forces General Hospital, Tao-Yuan, Taiwan.
authors.forename: Kuo-An
authors.initials: KA
authors.lastname: Wu
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Ming-Ling
authors.initials: ML
authors.lastname: Kuo
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Gin-Wen
authors.initials: GW
authors.lastname: Chang
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. Electronic address: hhlin@mail.cgu.edu.tw.
authors.forename: Hsi-Hsien
authors.initials: HH
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012190:Retroviridae / Q000378:metabolism; D012995:Solubility
chemical_list: C118925:ADGRG1 protein, human; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins
keywords: Adhesion-class G protein-coupled receptor; ELISA; GPR56; Soluble GPR56