Large-scale production and protein engineering of G protein-coupled receptors for structural studies

Milić, Dalibor; Veprintsev, Dmitry B.

doi:10.3389/fphar.2015.00066

Cited by 48 publications

(35 citation statements)

References 250 publications

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“…Second, despite the recent progress in the cloning and expression of GPCR proteins in cells, their expression levels vary greatly, hampering an effective strategy to study these proteins in parallel and on a genomic scale 5 . Third, a considerable number of GPCRs are toxic to the host cells once overexpressed, making it difficult to establish stable cell lines that constitutively express these receptors 6 . Finally and perhaps most importantly, 116 (31%) of the ~370 non-odorant GPCRs are orphans, without known physiological agonists or antagonists, which precludes study of their activity.…”

Section: Introductionmentioning

confidence: 99%

Development and Application of a High-Content Virion Display Human GPCR Array

Syu

Wang

et al. 2018

Preprint

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G protein-coupled receptors (GPCRs) comprise the largest membrane protein family in humans and can respond to a wide variety of ligands and stimuli. Like other multi-pass membrane proteins, the biochemical properties of GPCRs are notoriously difficult to study because they must be embedded in lipid bilayers to maintain their native conformation and function. To enable an unbiased, high-throughput platform to profile biochemical activities of GPCRs in native conformation, we individually displayed 315 human non-odorant GPCRs (>85% coverage) in the envelope of human herpes simplex virus-1 and immobilized on glass to form a high-content Virion Display (VirD) array. Using this array, we found that 50% of the tested commercial anti-GPCR antibodies (mAbs) is ultra-specific, and that the vast majority of those VirD-GPCRs, which failed to be recognized by the commercial mAbs, could bind to their canonical ligands, indicating that they were folded correctly. Next, we used the VirD-GPCR arrays to examine binding specificity of two known peptide ligands and recovered expected interactions, as well as new off-target interactions, three of which were confirmed with real-time kinetics measurements. Finally, we explored the possibility of discovering novel pathogen targets by probing VirD-GPCR arrays with live group B Streptococcus (GBS), a commonGram-positive bacterium causing neonatal meningitis. Using cell invasion assays and a mouse model of hematogenous meningitis, we showed that inhibition of one of the five newly identified GPCRs, CysLTR1, greatly reduced GBS penetration in brain-derived endothelial cells and in mouse brains. Therefore, our work demonstrated that the VirD-GPCR array holds great potential for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and deorphanization.

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Section: Introductionmentioning

confidence: 99%

Development and Application of a High-Content Virion Display Human GPCR Array

Syu

Wang

et al. 2018

Preprint

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“…B). When compared to previous studies, this was the most successful case of high cell density cultivation for GPCR production in E. coli (Milic and Veprintsev, ).…”

Section: Resultsmentioning

confidence: 85%

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

Lee

et al. 2016

Biotech & Bioengineering

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Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

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“…4B and 4C). 31 P NMR was also recorded to determine the presence of seven phosphorylation sites in peptide 1 (Fig. 4D and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Our model peptide for this study was derived from the C-tail of rhodopsin (Rho), a light sensitive G protein-coupled receptor (GPCR). [29][30][31] The C-terminal domain of rhodopsin protein (Rho330-348, DDEASTTVSKTETSQVAPA) becomes highly phosphorylated at Ser and Thr residues (underlined) during light activation, leading to an increased affinity of arrestin towards the receptor and inhibiting further G protein signalling. [32][33][34] Synthetic and homogeneous multi-phosphorylated peptides derived from this domain are essential tools to study the specific impact of each phosphorylation site or pattern on the regulatory function of rhodopsin.…”

Section: Introductionmentioning

confidence: 99%

A combinatorial approach for the synthesis of multi-phosphorylated peptides: new tool for studying phosphorylation patterns in proteins

Samarasimhareddy

Mayer

Metanis

et al. 2017

Preprint

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Phosphorylation of proteins at multiple sites creates different phosphorylation patterns that are essential for their biological activity. For example, such patterns contribute to the redirection of signalling to alternative pathways. Multi-phosphorylated peptides are excellent tools to systematically study the impact of unique phosphorylation patterns on signalling, but their synthesis is extremely difficult. Here we present an efficient and general method for the synthesis of multi-phosphorylated peptides, using a combination of different tailor-made coupling conditions. The method was demonstrated for the synthesis of a library of Rhodopsin C terminal peptides with distinct phosphorylation patterns containing up to seven phosphorylated Ser (pSer) and Thr (pThr) residues in close proximity to one another. Our method can be used to synthesize peptides incorporating multiple phosphorylated amino acids at high efficiency. It does not require any special expertise and can be performed in any standard peptide laboratory. This approach opens the way for quantitative mechanistic studies of phosphorylation patterns and their biological roles.

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Large-scale production and protein engineering of G protein-coupled receptors for structural studies

Cited by 48 publications

References 250 publications

Development and Application of a High-Content Virion Display Human GPCR Array

Development and Application of a High-Content Virion Display Human GPCR Array

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

A combinatorial approach for the synthesis of multi-phosphorylated peptides: new tool for studying phosphorylation patterns in proteins

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