The first use of the two-hybrid system is attributed to Song and Fields (1989). Roger Brent's lab (Harvard University, Boston) has developed a nice variant of the system
and he has a lot of www-information on two hybrid screens, and Stephen J. Elledge's lab (Baylor College of Medicine, Houston) has contributed a lot to the development of useful systems.
Interaction trap cloning with yeast.
A draft manuscript by Russell L. Finley Jr. and Roger Brent to appear in "Gene Probes - A practical Approach" published by Oxford University Press. Here are many protocols and tidbits on the methods.
Information on yeast strains in use.
Applications of interaction traps/ two hybrid systems to biotechnology research
This is a draft version of a manuscript by Andrew R. Mendelsohn and Roger Brent that is to appear in the October 1994 issue of Current Opinions in Biotechnology. It reviews the use of interaction technology in biotechnology.
Other useful stuff:
The Saccharomyces Information Resource and the Saccharomyces Genome Database at Stanford.
The yeast virtual library
Yeast cloning vectors
The BioSci Newsgroup for yeast. or search in the BioSci USENET archives.
A collection of LexA fusion plasmids (Erica Golemis, Fox Chase Cancer Center, N.J.)
Recovery of plasmids from yeast (method for S. pombe - but mentions E. coli JA226) Fission Yeast Handbook by Frans Hochstenbach, (University of Amsterdam).
Tabulation of typical false positives obtained in two-hybrid screens - and here is the LONG LIST (Erica Golemis, Fox Chase Cancer Center, N.J.).
pAS2 bait vector (gb:U30496)
Note: this list was scanned from the Clontech booklet devoted to the two-hybrid system; it was compiled in Oct. 1995. Some corrections were made, and some references were added.
Find the abstracts in MEDLINE
Some details of the two-hybrid system usage can be found in the Table 2 (NOT a comprehensive study)
Some data concerning false positives are available
Two-hybrid systems are extremely powerful methods for detecting protein:protein interactions in vivo. The basis of two-hybrid systems is the modular domains found in some transcription factors. In the CheckMate Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA binding domain upstream of a multiple cloning region, and the pACT Vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. In addition, the pBIND Vector expresses the Renilla reniformis luciferase, which allows the user to normalize the transfection efficiency. The two genes encoding the two potentially interactive proteins of interest are cloned into pBIND and pACT Vectors to generate fusion proteins with the DNA binding domain of GAL4 and the activation domain of VP16, respectively. The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn, is upstream of the firefly luciferase gene (luc+). The pGAL4 and pVP16 fusion constructs are transfected along with pG5luc Vector into mammalian cells. Two to three days after transfection, the cells are lysed, and the amounts of Renilla luciferase and firefly luciferase are quantitated using the Dual-Luciferase® Reporter Assay System. Interaction between the two test proteins, as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression over the negative controls. Two positive control vectors are provided: pBIND-Id and pACT-MyoD Control Vectors, containing GAL4:Id and VP16:MyoD, respectively.
Screen cDNA libraries for novel proteins that interact with a known bait protein or a known DNA sequence, confrim interactions between know proteins
Description
CLONTECH's MATCHMAKER product line is a comprehensive set of tools for studying protein interactions through two-hybrid, one-hybrid, and related assays.
The MATCHMAKER Two-Hybrid System 3 is an enhanced GAL4 two-hybrid system that uses three different reporter genes with distinct control regions to virtually eliminate false positives. The pBridge Vector can be used to create a three-hybrid system to screen for interactions involving three proteins. For proteins that are cytotoxic or cytostatic in yeast, the MATCHMAKER LexA Two-Hybrid System uses inducible promoters so the DNA-BD- and AD-fusion proteins are only expressed during the actual screen. CLONTECH also provides the Mammalian MATCHMAKER Two-Hybrid Assay Kit to verify interactions in a mammalian system. Finally, the MATCHMAKER One-Hybrid System reveals protein interactions with DNA sequences.
CLONTECH also provides high-quality MATCHMAKER cDNA Libraries from a broad range of tissues and species for both the GAL4- and LexA-based two-hybrid systems.
Hybrigenics is the functional proteomics company dedicated to the identification of novel biological targets involved in key cellular functions, using protein-protein interactions to discover lead compounds which are the precursors of future medicines.
The increasing pressure on the pharmaceutical industry to lower cost, and accelerate the discovery and marketing of innovative therapeutics is well-recognized. In the context of gene-based drug discovery, functional proteomics is well adapted to fill the gap between structural genomic information and biological function.
Using genomic DNA from microbial pathogens or cDNA from normal or diseased tissues, Hybrigenics first identifies interacting proteins, then reconstitutes functional signaling or metabolic pathways, and finally identifies modulators (inhibitors or upregulators) of these interactions. These modulators may constitute lead compounds for the development of novel pharmaceuticals.
Hybrigenics offers four types of Products:
1 - Protein Interaction Maps: PIMs® for whole microbial pathogen genomes or selected cDNA libraries. First PIMs® available:
Helicobacter pylori (example below)
Hepatitis C Virus
Saccharomyces cerevisiae
2 - Proprietary selected drug targets:
Additional information is available upon request for the following programs:
Helicobacter pylori
Hepatitis C Virus
Saccharomyces cerevisiae
3 - Small molecule lead compounds:
Helicobacter pylori: SIDs®
Hepatitis C Virus: SIDs®
Saccharomyces cerevisiae: SIDs®
Hefe Two-Hybrid Systeme
Das Yeast Two-Hybrid System ist ein recht junges Verfahren zur Detektion von Protein-Protein-Wechselwirkungen in vivo ("Interaction Trap").
Das Verfahren wurde von Fields und Song entwickelt und ist mittlerweile bei einigen Firmen als "Kit" kommerziell erhältlich (z.B. Clontech, Invitrogen). Im Folgenden sind einige Links aufgeführt, die Einführungen zu diesem Thema oder einige nützliche Tips enthalten. Außerdem ibt es ein paar Links zu allgemeinen Hefe Genetik.
