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Protein Expr Purif. 2015 May;109:85-92. doi: 10.1016/j.pep.2014.11.013. Epub 2014 Nov 28.

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

Author information

1
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
2
Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
3
Division of Rheumatology, Allergy and Immunology, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan.
4
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.
5
Department of Internal Medicine, Taoyuan Armed Forces General Hospital, Tao-Yuan, Taiwan.
6
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.
7
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.
8
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.

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.

KEYWORDS:

Adhesion-class G protein-coupled receptor; ELISA; GPR56; Soluble GPR56

PMID:
25437104
DOI:
10.1016/j.pep.2014.11.013
[Indexed for MEDLINE]

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