A tetracycline repressor-based mammalian two-hybrid system to detect protein–protein interactions in vivo

Thibodeaux, Gabrielle Nina; Cowmeadow, Roshani B.; Umeda, Aiko; Zhang, Zhiwen

doi:10.1016/j.ab.2008.11.042

Cited by 9 publications

(8 citation statements)

References 20 publications

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“…We first tested the interaction of TOP1 and BOT1 to the Abl SH3 domain in vivo using the established tetracycline repressor-based mammalian two hybrid system (trM2H) that is sensitive to weak interactions [20]. This system takes advantages of the dimeric TetR transcription regulator which binds to the Tet-responsive element (TRE) sequence at upstream of reporter gene (AcGFP) to induce its expression.…”

Section: Resultsmentioning

confidence: 99%

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A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker

Umeda

Thibodeaux²,

Moncivais³

et al. 2010

Analytical Biochemistry

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The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bio-analytical reagents. One approach to this problem is to incorporate a redox-active amino acid 3,4-dihydroxy-L-phenylalanine (L-DOPA) which selectively forms a covalent linkage to the target protein. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide TOP1 that weakly binds to the target protein, the SH3 domain of human Abl kinase, was fused to green fluorescent protein (GFP) and L-DOPA was site-specifically incorporated into the peptide region (TOP1-DOPA-GFP). TOP1-DOPA-GFP produced from E. coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.

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Section: Resultsmentioning

confidence: 99%

“…Restriction enzyme sites AgeI and KpnI were introduced into pTetOff previously to afford pTOAgeIKpnI [20]. The coding sequences for TOP1 and BOT1 were purchased as complementary oligonucleotides (Invitrogen) and annealed.…”

Section: Methodsmentioning

confidence: 99%

A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker

Umeda

Thibodeaux²,

Moncivais³

et al. 2010

Analytical Biochemistry

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“…Recent studies have demonstrated that the C-terminal catalytic domain of SrtA selectively forms a homo-dimer in vitro and in E. coli. [18][19][20][21] Herein, we demonstrate that SrtA also forms homo-dimer on the membrane of S. aureus and such dimerization mediates SrtA's activities on the cell membrane, suggesting that the dimerization of SrtA is one of the mechanisms to globally coordinate the cell surface decoration on S. aureus.…”

Section: Introductionmentioning

confidence: 95%

Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

Zhu

Xiang

Jiang

et al. 2015

Exp Biol Med (Maywood)

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Staphylococcus aureus sortase A (SrtA) transpeptidase is a therapeutically important membrane-bound enzyme in Gram-positive bacteria, which organizes the covalently attached cell surface proteins on the peptidoglycan cell wall of the organism. Here, we report the direct observation of the highly selective homo-dimerization of SrtA on the cell membrane. To address the biological significance of the dimerization towards enzyme function, site-directed mutagenesis was performed to generate a SrtA mutant, which exists as monomer on the cell membrane. We observed that the cell surface display of adhesive proteins in S. aureus cells expressing monomeric SrtA mutant is more prominent than the cells expressing the wild-type enzyme. A cell-based invasion assay was also performed to evaluate the activities of wild-type SrtA and its monomeric mutant as well. Our data demonstrated that S. aureus cells expressing SrtA in monomeric form invade host mammalian cells more efficiently than those expressing wild-type SrtA in dimer-monomer equilibrium. The results suggested that the monomeric form of SrtA is more active than the dimeric form of the enzyme in terms of cell surface display of virulence factors for infection. This is the first study to present the oligomerization of SrtA and its related biological function on the cell membrane. Study of SrtA dimerization has implications for understanding its catalytic mechanism at the cellular level as well as the development of novel anti-infective agents.

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“…Another variation on the two-hybrid theme in mammalian cells is the tetracycline repressor-based system, trM2H (638). In trM2H, hybrids are composed of three functional parts: DNA binding of two tetracycline repressor (TetR) fragments is restored upon bait-prey interaction, leading to activation of transcription by the VP16 AD domain.…”

Section: Mammalian Genetic Protein-protein Interaction Systemsmentioning

confidence: 99%

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Stynen

Tournu

Tavernier

et al. 2012

Microbiol Mol Biol Rev

178

143

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SUMMARY The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli , but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays.

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A tetracycline repressor-based mammalian two-hybrid system to detect protein–protein interactions in vivo

Cited by 9 publications

References 20 publications

A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker

A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker

Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

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