After 5 min of incubation, the luminescence intensity was measured using a Lucy-2 luminometer (Aloka, Tokyo, Japan). 4.4.2. 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 Apalutamide (ARN-509) TRAb immunoassay in GD patients. Keywords: thyroid-stimulating hormone receptor, AMB-1, autoantibody, bacterial magnetic particles, tetracycline-inducible expression system 1. Introduction 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]. The stimulation of autoantibodies to TSHR (TRAbs) is known to be associated with hyperthyroidism in Graves disease (GD), and measurement of TRAbs is important for diagnosis of GD [2,3]. Currently available immunoassays for measuring TRAb are competitive radioimmunoassay using I125-labelled TSH or enzyme-linked immunosorbent assay (ELISA) using biotin-labeled TSH (TSH-biotin) [4,5]. More recently, Rabbit polyclonal to pdk1 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. Given that 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 Apalutamide (ARN-509) of human TSHR (hTSHR) in current TRAb immunoassays. However, the use of thyroid membrane extract carries with it the potential for lot-to-lot inconsistency, and differences in species may influence the detection of autoantibodies to hTSHR [7]. To avoid these possible risks, the development of TRAb assay using recombinant hTSHR is desirable. AMB-1 is a gram-negative, facultative anaerobic bacterium that is known to produce bacterial magnetic particles (BacMPs) which form a magnetosome chain in the cytoplasm under anaerobic conditions. BacMPs, which are typically 50C100 nm in size, consist of magnetite (Fe3O4) surrounded by a lipid bilayer membrane, and exhibit strong ferrimagnetism. Several membrane-integrated or tightly bound proteins are known to be abundant on the surface of BacMPs [8]. Using these characteristics, we have succeeded to date in functionally displaying soluble proteins on BacMPs by gene fusion techniques, using either MagA, Mms16, or Mms13 as an anchor molecule, with applications in purposes such as immunoassay, enzyme reaction, ligand-receptor interaction or cell separation [9C12]. The main advantage of the BacMP-based expression system is that the protein of interest is easily and directly isolated using a magnet. We recently applied these techniques to overexpress transmembrane proteins such as D1 dopamine Apalutamide (ARN-509) 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]. However, applications of transmembrane proteins, especially of GPCRs, are currently limited. Here we describe the successful expression of Mms13-anchored full-length hTSHR in AMB-1 using a tetracycline-inducible expression system, and demonstrations of its ligand and autoantibody-binding activity. 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. 2. Results 2.1. Growth of hTSHR Transformants in a Tetracycline-Inducible Expression System We have previously observed extremely low expression of Mms13-hTSHR on BacMPs due to growth inhibition when constitutively Apalutamide (ARN-509) overexpressed in AMB-1 (data not shown). Accordingly, we investigated the use of a tetracycline-inducible expression system [13]. AMB-1 transformants harboring pUMtOR13TSHR (see Experimental section) were grown in magnetic spirillum growth medium (MSGM) with or without addition of anhydrotetracycline (ATc). 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). Likewise, the hTSHR transformant, but not wild-type AMB-1, underwent significant growth inhibition after the addition of ATc at mid-log phase (Figure 1). These results indicate that expression of Mms13-hTSHR is toxic to AMB-1, and that inducible expression is necessary. Open in a separate window Figure 1 Growth curves of the AMB-1 transformant of pUMtOR13TSHR. The transformant was grown in magnetic spirillum growth medium (MSGM) with or without ATc. ATc (500 ng/mL) was added to the medium at the time of inoculation (filled circle) or at mid-log phase, indicated by solid arrow (open triangles). Open squares show growth curves in the absence of ATc. 2.2. Isolation of hTSHR-Displaying BacMPs Figure 2a shows the procedure for isolation of hTSHR-displaying BacMPs. 6.5 mg of BacMPs were isolated from a 5 L culture of AMB-1 transformants of Mms13-hTSHR after induction with ATc. Inducible expression of the Mms13-hTSHR fusion protein on BacMPs was evaluated by ELISA using anti-hTSHR antibody. As shown in Figure 2b, low-level expression of Mms13-hTSHR was observed without induction of ATc. On the other hand, an approximately 9-fold increase in the expression of Mms13-hTSHR was detected on BacMPs in transformants after ATc induction (Figure 2b), demonstrating that inducible expression system is effective for hTSHR expression on BacMPs. Open in a separate window Figure 2 Confirmation of hTSHR expression on BacMPs. (a) Schematic diagram for preparation of BacMPs displaying hTSHR. Plasmids pUMtOR13TSHR.