Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems hav...

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Published inInternational journal of molecular sciences Vol. 20; no. 14; p. 3479
Main Authors Pedelacq, Jean-Denis, Cabantous, Stéphanie
Format Journal Article
LanguageEnglish
Published Switzerland MDPI AG 15.07.2019
MDPI
Subjects
Animals
Biochemistry, Molecular Biology
Biotechnology
E coli
Energy transfer
Engineering
Evolution
Experiments
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Green Fluorescent Proteins - standards
Humans
Life Sciences
Localization
Microscopy, Fluorescence - methods
Mutagenesis
Mutation
Neurosciences
Protein Engineering - methods
Protein Folding
Proteins
Review
Sensors
fluorescent protein
tripartite
folding
PPI
split-GFP
bipartite
superfolder
Online AccessGet full text

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Abstract Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.
AbstractList Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.
Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and expression of green fluorescent protein (GFP) color variants in the host organism have already been the subject of several reviews [1,2,3,4] and will not be described here. The auto-catalytic post-translational modifications of Ser 65, Tyr 66, and Gly 67 can occur when the protein is expressed in prokaryotic or eukaryotic species [8]. avGFP has a maximum excitation peak at 396–398 nm (corresponding to the neutral state of Tyr 66 in the chromophore) and a lower secondary peak at 476–478 nm (corresponding to the deprotonated anionic state of Tyr 66) [9,10], the emission peak is at 510 nm [9]. [...]the excitation of avGFP by the 488 nm line of the Argon laser (standard in microscopy and flow cytometry) indicates low fluorescence intensity levels. avGFP was then evolved by site-directed mutagenesis to optimize brightness and excitation efficiency at 488 nm. Subsequently, the gene sequence of the corresponding S65T/F64L double mutant was converted to human codons to form the “enhanced-GFP” (eGFP), which combines a high expression rate in mammalian cells and a fluorescence intensity 30 times higher than that of avGFP [12]. The advantage with bright cyan FPs donors is that they can accommodate a large palette of acceptors, either green, yellow, or orange, with high Förster resonance energy transfer (FRET) efficiencies. sfTq2 is now used in FRET experiments with acceptor mNeonGreen [32] for the detection of protein–protein interactions in cytoplasmic and periplasmic compartments. sfTq2 mutant C70V named sfTq2ox, when paired with mNeonGreen shows even brighter fluorescence signal in the periplasm [31].
Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.
Author Cabantous, Stéphanie
Pedelacq, Jean-Denis
AuthorAffiliation 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France
2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France
AuthorAffiliation_xml – name: 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France
– name: 2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France
Author_xml – sequence: 1
  givenname: Jean-Denis
  surname: Pedelacq
  fullname: Pedelacq, Jean-Denis
– sequence: 2
  givenname: Stéphanie
  orcidid: 0000-0002-8406-9421
  surname: Cabantous
  fullname: Cabantous, Stéphanie
BackLink https://www.ncbi.nlm.nih.gov/pubmed/31311175$$D View this record in MEDLINE/PubMed
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Keywords fluorescent protein
tripartite
folding
PPI
split-GFP
bipartite
superfolder
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Snippet Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of...
Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and...
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SubjectTerms Animals
Biochemistry, Molecular Biology
Biotechnology
E coli
Energy transfer
Engineering
Evolution
Experiments
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Green Fluorescent Proteins - standards
Humans
Life Sciences
Localization
Microscopy, Fluorescence - methods
Mutagenesis
Mutation
Neurosciences
Protein Engineering - methods
Protein Folding
Proteins
Review
Sensors
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Title Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology
URI https://www.ncbi.nlm.nih.gov/pubmed/31311175
https://www.proquest.com/docview/2333580166
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https://pubmed.ncbi.nlm.nih.gov/PMC6678664
Volume 20
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