Abstract
The measurement of autoantibodies to thyroid-stimulating hormone receptor (TSHR) is important for the diagnosis of autoimmune thyroid disease such as Graves’ disease (GD). Although TSHR from porcine thyroid membrane is commonly used for the measurement of TSHR autoantibodies (TRAb), recombinant human TSHR (hTSHR) remains ideal in terms of stable supply and species identity. Here we set out to express recombinant hTSHR on the lipid-bilayer surface of magnetic nanoparticles from a magnetotactic bacterium, Magnetospirillum magneticum AMB-1. Using a tetracycline-inducible expression system, we successfully overexpressed functional hTSHR on bacterial magnetic particles (BacMPs) in AMB-1 via an anchor protein specific for BacMPs. The overexpressed hTSHR was membrane integrated and possessed both ligand and autoantibody binding activity. Our data suggest that hTSHR-displayed BacMPs have potential as novel tools for ligand-receptor interaction analysis or for TRAb immunoassay in GD patients.
Highlights
Thyroid-stimulating hormone receptor (TSHR) belongs to the subfamily of rhodopsin-like members of the G-protein coupled receptor (GPCR) superfamily, and plays a central role in thyroid hormone production and regulation [1]
Given that thyroid-stimulating hormone receptor (TSHR), like other typical GPCRs, is notoriously difficult to overexpress in a soluble form, detergent-extracted porcine thyroid membrane is generally used as a source of TSHR instead of human TSHR in current TSHR autoantibodies (TRAb) immunoassays
When ATc was added at the start of inoculation, no growth of the TSHR transformant was observed, which was consistent with the previous result of constitutive expression (Figure 1)
Summary
Thyroid-stimulating hormone receptor (TSHR) belongs to the subfamily of rhodopsin-like members of the G-protein coupled receptor (GPCR) superfamily, and plays a central role in thyroid hormone production and regulation [1]. A biotin-labeled human monoclonal thyroid stimulating antibody, M22, has been used for TRAb ELISA instead of TSH-biotin [6] In these assays, preparation of functional TSHR protein is a critical step. The main advantage of the BacMP-based expression system is that the protein of interest is and directly isolated using a magnet We recently applied these techniques to overexpress transmembrane proteins such as D1 dopamine receptor, a member of the GPCR family, and a truncated form of CD81, a tetraspanin receptor for Hepatitis C Virus, and demonstrated ligand-binding activity [10,13]. This study raises the possibility of applications using hTHSR-displayed BacMPs for the analysis of ligand or autoantibody-receptor interaction, or for automated TRAb immunoassay
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