WO2009088991A3 - Flow cytometric gfp-based yeast two hybrid system - Google Patents

Flow cytometric gfp-based yeast two hybrid system Download PDF

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Publication number
WO2009088991A3
WO2009088991A3 PCT/US2009/000043 US2009000043W WO2009088991A3 WO 2009088991 A3 WO2009088991 A3 WO 2009088991A3 US 2009000043 W US2009000043 W US 2009000043W WO 2009088991 A3 WO2009088991 A3 WO 2009088991A3
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WO
WIPO (PCT)
Prior art keywords
protein
reporter
screening
flow cytometric
liquid culture
Prior art date
Application number
PCT/US2009/000043
Other languages
French (fr)
Other versions
WO2009088991A2 (en
Inventor
Hong Cai
Jun Chen
Original Assignee
Los Alamos National Security, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Los Alamos National Security, Llc filed Critical Los Alamos National Security, Llc
Publication of WO2009088991A2 publication Critical patent/WO2009088991A2/en
Publication of WO2009088991A3 publication Critical patent/WO2009088991A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Abstract

The invention provides an improved yeast two-hybrid screening system that is optimal for high throughput screening of cDNA libraries for protein-protein interactions. The invention utilizes a combination of stably-integrated yEGFP as a reporter in the reporter yeast strain, combined with flow cytometric detection of fluorescence resulting from transcriptional activation of the reporter protein following interaction of protein-protein pairs. The system of the invention maybe conducted using liquid culture, rather than the agar plating required in conventional Y2H systems, thereby saving substantial time and resources. Moreover, by virtue of the system's liquid culture approach the invention is adaptable to high-throughput format and can be used for large-scale cDNA screening for interacting proteins with high sensitivity and low signal:noise ratios. The invention's Y2H assay/system offers convenient, quantitative and faster reporter analysis compatible with existing liquid handling robots, and also reduces labor requirements in screening cDNA libraries.
PCT/US2009/000043 2008-01-03 2009-01-05 Flow cytometric gfp-based yeast two hybrid system WO2009088991A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1008508P 2008-01-03 2008-01-03
US61/010,085 2008-01-03

Publications (2)

Publication Number Publication Date
WO2009088991A2 WO2009088991A2 (en) 2009-07-16
WO2009088991A3 true WO2009088991A3 (en) 2009-12-30

Family

ID=40853400

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2009/000046 WO2009088994A1 (en) 2008-01-03 2009-01-05 Surface display-based yeast two hybrid screening system
PCT/US2009/000043 WO2009088991A2 (en) 2008-01-03 2009-01-05 Flow cytometric gfp-based yeast two hybrid system

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2009/000046 WO2009088994A1 (en) 2008-01-03 2009-01-05 Surface display-based yeast two hybrid screening system

Country Status (1)

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WO (2) WO2009088994A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590528A (en) * 2012-01-18 2012-07-18 厦门大学 Method for detecting interaction of proteins

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562576B2 (en) * 2001-01-04 2003-05-13 Myriad Genetics, Inc. Yeast two-hybrid system and use thereof
WO2005093086A1 (en) * 2004-03-16 2005-10-06 Cytomics Systems Method for screening agents modulating a lkb$g(a) protein ubiquitination and means for carrying out said method

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5491068A (en) * 1991-02-14 1996-02-13 Vicam, L.P. Assay method for detecting the presence of bacteria
WO2006026712A2 (en) * 2004-08-31 2006-03-09 Fox Chase Cancer Center Yeast/bacterial two-hybrid system and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562576B2 (en) * 2001-01-04 2003-05-13 Myriad Genetics, Inc. Yeast two-hybrid system and use thereof
WO2005093086A1 (en) * 2004-03-16 2005-10-06 Cytomics Systems Method for screening agents modulating a lkb$g(a) protein ubiquitination and means for carrying out said method

Also Published As

Publication number Publication date
WO2009088991A2 (en) 2009-07-16
WO2009088994A1 (en) 2009-07-16

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