The Expression of Soluble, Full-Length, Recombinant Human TSH Receptor in a Prokaryotic System

Busuttil, Bridget E.; Turney, Kerrin L.; Frauman, Albert G

doi:10.1006/prep.2001.1519

Cited by 14 publications

(4 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using bacterially-expressed hTSHR ectodomain, some groups have demonstrated ligand binding activity either in a native form [15], or reactivity toward serum autoantibody of a denatured form [16], although not all have been successful [17–19]. Soluble, full-length hTSHR expression has been reported in a prokaryotic system [20], in which immunoreactivity to sera in two GD patients was shown by Western blotting. It has remained unknown, therefore, whether the native form of bacteria-specific hTSHR has ligand or autoantibody binding activity.…”

Section: Discussionmentioning

confidence: 99%

Functional Expression of Thyroid-Stimulating Hormone Receptor on Nano-Sized Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Sugamata

Uchiyama

Honda

et al. 2013

IJMS

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Functional Expression of Thyroid-Stimulating Hormone Receptor on Nano-Sized Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Sugamata

Uchiyama

Honda

et al. 2013

IJMS

View full text Add to dashboard Cite

show abstract

“…This kind of difference suggests that the behaviour in terms of expression level is clearly receptor dependent. As shown in table 2, expression levels range from 0.2 to 16 pmol of receptor per milligram of membrane protein (equivalent to 15 -450 sites per cell), and are quite low whatever the strategy employed (with the notable exception of the TSH receptor, see [126]). Moreover, within a given receptor family the results can be different even though the expression strategy employed is identical.…”

Section: Main Characteristics Of E Colimentioning

confidence: 97%

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Sarramegna¹,

Talmont²,

Demange³

et al. 2003

CMLS, Cell. Mol. Life Sci.

216

201

View full text Add to dashboard Cite

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

show abstract

“…Microbial organisms, such as the bacterium Escherichia coli , have been extensively used for the expression of MPs for structural studies (Wagner et al 2006) given their ease of growth and manipulation, and ability to be propagated in chemically defined medium, allowing for substitution with selenomethionine for X‐ray crystal structure determination (Hendrickson et al 1990) or with isotopically labeled amino acids for NMR structural studies. However, when expressed in E. coli , GPCRs are often toxic to the host cell, are subject to degradation, or accumulate in inclusion bodies that, with some exceptions (Busuttil et al 2001;, Baneres et al 2003;, 2005; Tian et al 2005), are difficult to solubilize and refold (Lundstrom et al 2006). Only a very limited number of human class I GPCR, such as the peripheral cannabinoid receptor and the neurotensin receptor, fused to polypeptides that prevent aggregation or enhance stability, have been expressed in membrane‐integrated form in E. coli (Tucker and Grisshammer 1996; Calandra et al 1997).…”

mentioning

confidence: 99%

Efficient production of membrane‐integrated and detergent‐soluble G protein‐coupled receptors in Escherichia coli

et al. 2008

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.Keywords: G protein-coupled receptor; Escherichia coli; membrane protein; FtsH; genetic engineering; cannabinoid receptor; bradykinin receptor Supplemental material: see www.proteinscience.orgThe array of functions that membrane proteins (MPs) perform in cells is very broad, ranging from the maintenance of cell structure and protection against toxins in the environment to the production of energy and respiration. Consistent with these vital roles, genes for MPs typically account for 15%-30% of an organism's genome. However, <0.5% of the protein structures contained in the Protein Data Bank are of MPs. Furthermore, the great majority of extant MP structures are of prokaryotic proteins, with only about two dozen distinct eukaryotic MP structures currently available to the public (see http:// blanco.biomol.uci.edu/Membrane_Proteins_xtal.html for a well-curated database of known MP structures). The reason for this relative lag in structural biology efforts toward MPs is twofold: The first bottleneck is the determination of suitable crystallization conditions (Loll 2003). The second limitation is the production of sufficient amounts of MP suitable for initiation/optimization of crystallization trials. The latter limitation is particularly pertinent to mammalian MPs, which may be present 6 Present address:

show abstract

The Expression of Soluble, Full-Length, Recombinant Human TSH Receptor in a Prokaryotic System

Cited by 14 publications

References 28 publications

Functional Expression of Thyroid-Stimulating Hormone Receptor on Nano-Sized Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Functional Expression of Thyroid-Stimulating Hormone Receptor on Nano-Sized Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Efficient production of membrane‐integrated and detergent‐soluble G protein‐coupled receptors in Escherichia coli

Contact Info

Product

Resources

About