Escherichia coli One- and Two-Hybrid Systems for the Analysis and Identification of Protein–Protein Interactions

Hu, James C.; Kornacker, M. G.; Hochschild, Ann

doi:10.1006/meth.1999.0908

Cited by 94 publications

(91 citation statements)

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“…Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (9, 10), and in certain instances ablate protein function (11,12).Although the 2H system was initially developed by using yeast as a host organism, numerous bacterial (B)2H systems are now common laboratory tools and represent an experimental alternative with certain advantages over the yeast-based systems (13,14). A number of these bacterial approaches employ split activator/ repressor proteins; thus, they are functionally analogous to the GAL4-based yeast system (15-17).…”

mentioning

confidence: 99%

“…Although the 2H system was initially developed by using yeast as a host organism, numerous bacterial (B)2H systems are now common laboratory tools and represent an experimental alternative with certain advantages over the yeast-based systems (13,14). A number of these bacterial approaches employ split activator/ repressor proteins; thus, they are functionally analogous to the GAL4-based yeast system (15-17).…”

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confidence: 99%

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Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Waraho

DeLisa

2009

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a reliable genetic selection strategy for isolating interacting proteins based on the ''hitchhiker'' mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an Nterminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 ␤-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer ␤-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.ligand binding proteins ͉ protein folding quality control ͉ signal peptide ͉ twin-arginine translocation ͉ 2-hybrid system P rotein-protein interactions are key molecular events that integrate multiple gene products into functional complexes in virtually every cellular process. Because such interactions mediate numerous disease states and biological mechanisms underlying the pathogenesis of bacterial and viral infections, identification of protein-protein interactions remains one of the most important challenges in the postgenomics era. The yeast 2-hybrid (Y2H) system (1) has been the tool of choice for revealing numerous protein-protein interactions, underlying diverse protein networks and complex protein machinery inside living cells. To date, Y2H has been used to generate protein interaction maps for humans (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), Saccharomyces cerivisiae (5, 6), vaccinia virus (7), and Escherichia coli bacteriophage T7 (8). Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (9, 10), and in certain instances ablate protein functi...

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mentioning

confidence: 99%

mentioning

confidence: 99%

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Waraho

DeLisa

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…pAC cI-TrxA encodes cI residues 1-236 fused to three alanine residues, which, in turn, are fused to residues 2-109 of wild-type TrxA. cI-TrxA fusion vectors were constructed by cloning the appropriate NotI-BamHI-cut PCR products into NotI-BstYI-cut pAC cI32 (12). pAC⌬cI and pBR␣ have been described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…Expression of the hybrid ␣-thyA gene in pBR␣-ThyA is under the control of tandem lpp and lacUV5 promoters. The hybrid ␣-thyA gene was created by cloning the appropriate NotI-BamHI-cut PCR product into NotI-BamHI-cut pBR␣LN (12). Two-Hybrid Analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Aptamer targets could also be identified by using powerful genetic methods for detecting protein-protein interactions, termed two-hybrid screens (5,12,21,22). To test this concept, we measured the protein-protein interaction between ThyA and the TrxA101Q aptamer, using an E. coli-based, two-hybrid system (13,23).…”

Section: Two-hybrid Detection Of Interaction Between Thya and Aptamermentioning

confidence: 99%

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Isolation of peptide aptamers that inhibit intracellular processes

Blum

Dove

Hochschild

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible P BAD promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA ؊ phenotype that was suppressed by simultaneous overexpression of ThyA. Two-hybrid analysis showed that this aptamer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five such aptamers were characterized. Four aptamers were bacteriostatic when expressed, whereas one showed a bactericidal effect. Introduction of translational stop codons into various aptamers blocked their activity, suggesting that their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical tool for identifying targets for antibacterial drug development.

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Microbial Physiology in the Genomic Era: A Revolutionary Tale

Moat¹,

Foster²,

Spector³

2002

Microbial Physiology

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The field of microbial physiology is undergoing a dramatic revolution. This reformation is the direct result of three technological achievements: the personal computer, the Internet, and rapid DNA sequencing techniques. In the past, examining the physiology of a microorganism was a long, painstaking process. Even now, 60 years after initiating the analysis of the common intestinal microorganism Escherichia coli, we are still far from having a completely, integrated picture of its biochemistry and genetics.Today, however, many organisms that have proven almost intractable to scientific inquiry have had much of their genetics and physiology laid bare. Examples include Rickettsia prowazekii and Mycobacterium leprae, neither of which can grow outside of a living host. We now know with some certainty what biochemical pathways they have, how they make energy, what possible virulence proteins are in their pathogenic arsenal, and how they relate evolutionarily to other bacteria! All this has come about by modern sequencing techniques that have allowed entire genomes to be decoded in relatively short periods of time and because powerful sequence analysis software has been created that identifies open reading frames [an open reading frame (ORF) is a DNA sequence predicted to encode a protein] and promoter sequences, and compares new sequences with those already known. Comparing the amino acid sequence of an ORF of unknown function with all other known protein sequences (called a homology search) identifies those proteins with sequences and motifs similar to what is present in the ORF. This analysis provides considerable insight as to the possible function of the predicted ORF in the cell without ever having conducted a single, true biochemical experiment! The personal computer and Internet have fueled this renaissance of microbial physiology by allowing unfettered sharing of this information among scientists around the world.This chapter briefly describes the basic tools molecular biologists use to examine DNA sequences, gene expression, DNA-protein interactions, and protein-protein interactions. Initially, however, we discuss how the sequence of a complete genome is obtained and the means by which that knowledge can be used to study microbial physiology and evolution.

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Escherichia coli One- and Two-Hybrid Systems for the Analysis and Identification of Protein–Protein Interactions

Cited by 94 publications

References 49 publications

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Isolation of peptide aptamers that inhibit intracellular processes

Microbial Physiology in the Genomic Era: A Revolutionary Tale

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