In vivo characterisation of fluorescent proteins in budding yeast.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
19 02 2019
19 02 2019
Historique:
received:
15
10
2018
accepted:
14
12
2018
entrez:
21
2
2019
pubmed:
21
2
2019
medline:
4
9
2020
Statut:
epublish
Résumé
Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.
Identifiants
pubmed: 30783202
doi: 10.1038/s41598-019-38913-z
pii: 10.1038/s41598-019-38913-z
pmc: PMC6381139
doi:
Substances chimiques
Recombinant Proteins
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2234Références
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984-9
pubmed: 11050229
J Biol Chem. 2001 Aug 3;276(31):29188-94
pubmed: 11387331
Nat Biotechnol. 2002 Jan;20(1):87-90
pubmed: 11753368
Science. 2002 May 3;296(5569):913-6
pubmed: 11988576
Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82
pubmed: 12060735
Gene. 1992 Feb 15;111(2):229-33
pubmed: 1347277
Yeast. 2004 Jun;21(8):661-70
pubmed: 15197731
Nat Biotechnol. 2004 Dec;22(12):1567-72
pubmed: 15558047
Biochemistry. 2005 May 10;44(18):7085-94
pubmed: 15865453
Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13070-4
pubmed: 16135569
Nat Methods. 2005 Dec;2(12):910-9
pubmed: 16299476
Nat Biotechnol. 2006 Jan;24(1):79-88
pubmed: 16369541
Biochemistry. 2006 May 30;45(21):6570-80
pubmed: 16716067
Nat Methods. 2007 Jan;4(1):81-6
pubmed: 17179937
Biochemistry. 2007 Mar 27;46(12):3775-83
pubmed: 17323929
Nat Protoc. 2007;2(1):35-7
pubmed: 17401335
Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6672-7
pubmed: 17420458
Nat Methods. 2007 Jul;4(7):555-7
pubmed: 17572680
PLoS One. 2007 Oct 10;2(10):e1011
pubmed: 17925859
PLoS One. 2008 Apr 02;3(4):e1916
pubmed: 18382687
Nat Methods. 2008 Jun;5(6):545-51
pubmed: 18454154
Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9227-32
pubmed: 18574155
J Biol Chem. 2008 Oct 24;283(43):28980-7
pubmed: 18682399
Microbiology. 2009 Jan;155(Pt 1):268-78
pubmed: 19118367
Nat Methods. 2009 May;6(5):355-8
pubmed: 19363494
Nat Methods. 2010 Feb;7(2):137-9
pubmed: 20081836
Physiol Rev. 2010 Jul;90(3):1103-63
pubmed: 20664080
Biophys J. 2011 Aug 17;101(4):961-9
pubmed: 21843488
PLoS One. 2011;6(11):e27321
pubmed: 22114669
Traffic. 2012 May;13(5):643-9
pubmed: 22289035
Nat Commun. 2012 Mar 20;3:751
pubmed: 22434194
Nat Methods. 2012 Oct;9(10):1005-12
pubmed: 22961245
Genome Biol. 2012 Sep 10;13(9):R80
pubmed: 23021432
Nature. 2013 Mar 7;495(7439):111-5
pubmed: 23417067
Nat Methods. 2013 May;10(5):407-9
pubmed: 23524392
PLoS One. 2013 Jul 02;8(7):e67902
pubmed: 23844123
J Biotechnol. 2014 Dec 10;191:250-9
pubmed: 25107505
Methods Mol Biol. 2014;1205:45-78
pubmed: 25213239
Yeast. 2015 Apr;32(4):379-87
pubmed: 25612242
Mol Cell. 2015 Sep 3;59(5):744-54
pubmed: 26321254
Sci Rep. 2016 Feb 16;6:20889
pubmed: 26879144
Nat Methods. 2016 Jul;13(7):557-62
pubmed: 27240257
Mol Biol Cell. 2016 Nov 7;27(22):3385-3394
pubmed: 27385332
Sci Rep. 2016 Aug 03;6:30892
pubmed: 27485088
Nat Methods. 2017 Jan;14(1):53-56
pubmed: 27869816
J Gen Microbiol. 1987 Aug;133(8):2191-6
pubmed: 2832518
Bioinformatics. 2017 Aug 1;33(15):2424-2426
pubmed: 28369169
Sci Rep. 2017 Sep 20;7(1):11999
pubmed: 28931898
Sci Rep. 2017 Oct 24;7(1):13922
pubmed: 29066766
Cell Chem Biol. 2018 Mar 15;25(3):330-338.e7
pubmed: 29290624
Nat Methods. 2018 Jan;15(1):47-51
pubmed: 29320486
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501-4
pubmed: 7809066
Curr Biol. 1996 Feb 1;6(2):178-82
pubmed: 8673464
Nucleic Acids Res. 1996 Nov 15;24(22):4592-3
pubmed: 8948654
Biochemistry. 1997 Jun 3;36(22):6786-91
pubmed: 9184161
Nature. 1997 Jul 24;388(6640):355-8
pubmed: 9237752
J Biol Chem. 1998 Apr 3;273(14):8212-6
pubmed: 9525926
Annu Rev Biochem. 1998;67:509-44
pubmed: 9759496