A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to
Yeast 2 hybrid
affinity
computational biology
flow cytometry
fluorescence
protein-protein interactions
yeast
Journal
Molecular & cellular proteomics : MCP
ISSN: 1535-9484
Titre abrégé: Mol Cell Proteomics
Pays: United States
ID NLM: 101125647
Informations de publication
Date de publication:
04 2020
04 2020
Historique:
received:
22
07
2019
revised:
31
01
2020
pubmed:
6
2
2020
medline:
23
1
2021
entrez:
5
2
2020
Statut:
ppublish
Résumé
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.
Identifiants
pubmed: 32015065
pii: S1535-9476(20)35026-X
doi: 10.1074/mcp.TIR119.001692
pmc: PMC7124468
pii:
doi:
Substances chimiques
Peroxins
0
Proteome
0
Saccharomyces cerevisiae Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
701-715Informations de copyright
© 2020 Cluet et al.
Références
Mol Syst Biol. 2016 Apr 22;12(4):863
pubmed: 27107012
J Biol Chem. 2008 Mar 7;283(10):6136-44
pubmed: 18174172
J Biol Chem. 1996 Mar 22;271(12):6794-800
pubmed: 8636102
Nat Rev Genet. 2004 Feb;5(2):101-13
pubmed: 14735121
Nat Rev Mol Cell Biol. 2007 Jan;8(1):37-48
pubmed: 17183359
Cell. 1993 Nov 19;75(4):791-803
pubmed: 8242750
Gene. 1987;58(2-3):201-16
pubmed: 2828185
Microbiol Mol Biol Rev. 2012 Jun;76(2):331-82
pubmed: 22688816
Cell. 1990 Oct 5;63(1):133-9
pubmed: 2208277
FEBS Lett. 1972 Jan 15;20(1):47-52
pubmed: 11946379
J Mol Biol. 2008 Oct 10;382(3):721-33
pubmed: 18674545
Methods Enzymol. 2000;328:26-46
pubmed: 11075336
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13720-5
pubmed: 11106396
Methods Mol Biol. 2015;1278:409-31
pubmed: 25859966
Nat Methods. 2014 Dec;11(12):1203-6
pubmed: 25584376
Biotechniques. 2004 May;36(5):872-6
pubmed: 15152608
Nat Methods. 2017 Aug;14(8):819-825
pubmed: 28650476
J Biomol Screen. 2014 Apr;19(4):516-25
pubmed: 24476585
J Biol Chem. 2000 Apr 28;275(17):12515-20
pubmed: 10777539
Nat Rev Neurosci. 2001 Jun;2(6):408-16
pubmed: 11389474
Methods Enzymol. 2000;328:74-88
pubmed: 11075339
J Biol Chem. 2007 Sep 7;282(36):26503-16
pubmed: 17635919
J Biol Chem. 1997 Nov 21;272(47):29852-8
pubmed: 9368058
Curr Opin Biotechnol. 2006 Aug;17(4):387-93
pubmed: 16806892
Cytometry A. 2005 Mar;64(1):34-42
pubmed: 15700280
Methods Enzymol. 2017;586:333-358
pubmed: 28137570
Mol Cell Proteomics. 2008 Nov;7(11):2279-87
pubmed: 18622019
J Mol Biol. 1995 Apr 28;248(2):478-86
pubmed: 7739054
J Cell Sci. 1996 Jan;109 ( Pt 1):257-62
pubmed: 8834810
J Mol Biol. 1997 Apr 4;267(3):696-706
pubmed: 9126847
Biotechniques. 2005 Dec;39(6):814, 816, 818 passim
pubmed: 16382897
EMBO J. 2001 Jun 15;20(12):2995-3007
pubmed: 11406576
Elife. 2018 Aug 10;7:
pubmed: 30095407
Biochemistry. 1998 Oct 20;37(42):14683-91
pubmed: 9778343
Nat Protoc. 2006;1(3):1066-91
pubmed: 17406388
Cytometry A. 2008 Apr;73(4):312-20
pubmed: 18307271
Proc Natl Acad Sci U S A. 2017 Nov 14;114(46):12166-12171
pubmed: 29087945
Mol Cell Biol. 1994 Feb;14(2):923-33
pubmed: 8289832
Genetics. 2014 Apr;196(4):951-60
pubmed: 24451596
Nature. 1989 Jul 20;340(6230):245-6
pubmed: 2547163
Nat Methods. 2009 Jan;6(1):91-7
pubmed: 19060903
Cell. 1996 Mar 22;84(6):863-74
pubmed: 8601310
Nucleic Acids Res. 1997 Jan 15;25(2):451-2
pubmed: 9016579
Proc Natl Acad Sci U S A. 2007 May 15;104(20):8247-52
pubmed: 17494738
Nat Struct Biol. 1996 Mar;3(3):244-51
pubmed: 8605626
Nature. 2012 Sep 27;489(7417):585-9
pubmed: 22940862
Genet Mol Res. 2003 Mar 31;2(1):124-35
pubmed: 12917809
Biochemistry. 1999 Jan 5;38(1):11-21
pubmed: 9890878
Cytometry A. 2012 Jan;81(1):90-8
pubmed: 21954189
J Biol Chem. 2009 Nov 13;284(46):31893-902
pubmed: 19776012
Mol Cell Biol. 1995 Oct;15(10):5820-9
pubmed: 7565735
Mol Cell Biol. 1992 Jul;12(7):3006-14
pubmed: 1620111
Nature. 2000 Nov 16;408(6810):307-10
pubmed: 11099028
Science. 2009 Nov 27;326(5957):1220-4
pubmed: 19965465
J Biol Chem. 2002 Jun 21;277(25):22798-805
pubmed: 11934902
J Biol Chem. 2009 Jun 12;284(24):16369-76
pubmed: 19369257
Nat Protoc. 2007;2(1):1-4
pubmed: 17401330