Strategies to improve the fluorescent signal of the tripartite sfGFP system
© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissionsoup.com..
Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein-protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1-9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1-9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2020 |
|---|---|
| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:52 |
|---|---|
| Enthalten in: |
Acta biochimica et biophysica Sinica - 52(2020), 9 vom: 08. Sept., Seite 998-1006 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Shen, Jing [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
147336-22-9 |
|---|
| Anmerkungen: |
Date Completed 06.01.2021 Date Revised 06.01.2021 published: Print Citation Status MEDLINE |
|---|
| doi: |
10.1093/abbs/gmaa073 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM31161325X |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM31161325X | ||
| 003 | DE-627 | ||
| 005 | 20231225142627.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2020 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1093/abbs/gmaa073 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1038.xml |
| 035 | |a (DE-627)NLM31161325X | ||
| 035 | |a (NLM)32582951 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Shen, Jing |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Strategies to improve the fluorescent signal of the tripartite sfGFP system |
| 264 | 1 | |c 2020 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 06.01.2021 | ||
| 500 | |a Date Revised 06.01.2021 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissionsoup.com. | ||
| 520 | |a Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein-protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1-9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1-9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a bimolecular fluorescence complementation | |
| 650 | 4 | |a bipartite sfGFP | |
| 650 | 4 | |a protein–protein interaction | |
| 650 | 4 | |a sfGFP | |
| 650 | 4 | |a tripartite sfGFP | |
| 650 | 7 | |a Green Fluorescent Proteins |2 NLM | |
| 650 | 7 | |a 147336-22-9 |2 NLM | |
| 700 | 1 | |a Zhang, Wenlu |e verfasserin |4 aut | |
| 700 | 1 | |a Gan, Chunyang |e verfasserin |4 aut | |
| 700 | 1 | |a Wei, Xiafei |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Jie |e verfasserin |4 aut | |
| 700 | 1 | |a Sun, Yuxue |e verfasserin |4 aut | |
| 700 | 1 | |a Yuan, Yi |e verfasserin |4 aut | |
| 700 | 1 | |a Cai, Xuefei |e verfasserin |4 aut | |
| 700 | 1 | |a Long, Quanxin |e verfasserin |4 aut | |
| 700 | 1 | |a Cui, Jing |e verfasserin |4 aut | |
| 700 | 1 | |a Guo, Haitao |e verfasserin |4 aut | |
| 700 | 1 | |a Huang, Ailong |e verfasserin |4 aut | |
| 700 | 1 | |a Hu, Jieli |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Acta biochimica et biophysica Sinica |d 2004 |g 52(2020), 9 vom: 08. Sept., Seite 998-1006 |w (DE-627)NLM144395010 |x 1745-7270 |7 nnns |
| 773 | 1 | 8 | |g volume:52 |g year:2020 |g number:9 |g day:08 |g month:09 |g pages:998-1006 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1093/abbs/gmaa073 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 52 |j 2020 |e 9 |b 08 |c 09 |h 998-1006 | ||