Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 26;15(3):e0230344.
doi: 10.1371/journal.pone.0230344. eCollection 2020.

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

Constanze Stumpf  1 , Tobias Wimmer  1 , Birgit Lorenz  1 , Knut Stieger  1
Affiliations

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

Constanze Stumpf et al. PLoS One. .

Abstract

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

PubMed Disclaimer

Conflict of interest statement

The authors have read the journal's policy and have the following competing interests: The authors would like to declare the following patents/patent applications associated with this research: TW, KS and BL have a patent pending on the method for measurement and control of intraocular VEGF concentration (EP 3211422 A1, WO 2017/144416 pending). This does not alter our adherence to all the PLOS ONE policies on sharing data and materials

Figures

Fig 1
Fig 1. eBRET2 VEGF biosensors.
(A) VEGF quantification method of the eBRET2 VEGF biosensor. A conformational change after VEGF binding leads to an increased energy transfer and thus to a higher BRET signal, in dependence of the VEGF concentration. (B) Overlapping Rluc8 emission wavelength and GFP2 excitation wavelength generates GFP2 emission at higher wavelength. (C) Biosensor expression constructs Re01–Re10. CMV = cytomegalovirus Promoter, Rluc8 = Renilla luciferase, R = VEGF receptor, D = IgG domain, GFP2 = green fluorescent protein, pA = polyadenylation signal. VEGF receptor 1 (R1) and 2 (R2) structure. The receptors contain a transmembrane-, an intracellular catalytic domain and 7 extracellular IgG like domains. IgG like domain 2 (D2) and 3 (D3) are responsible for the VEGF binding and were introduced between the Rluc8 and the GFP2 as VEGF binding domain.
Fig 2
Fig 2. Characterization of eBRET2 biosensor expression.
(A) Fluorescence microscopy of GFP2 expression after biosensor plasmid transfection in HEK293 T cells. (B) Luciferase activity via Luminescence measurement. Rluc8 expression correlates with Luciferase activity. (C) eBRET2 ratios of the biosensor variants normalized with the correction factor (Rluc8 29.8 ± 0.3 mBU). Significance defined as p < 0.001. (D) Western Blot shows full length expression of the biosensors and correction factor (Rluc8 alone). 1. Antibody: anti Rluc8 (1:1000) on the top and anti GAPDH (1:1000) on the bottom. 2. Antibody: anti Rabbit HRP (1:20000). Arrow indicates additional bands that show extra splice variants. (E) Splice prediction with Human Splicing Finder 3.0 software. Circles represent donor sites, squares represent acceptor sites, and strongest candidates are located in the grey area. Arrow marked the strong donor splice site in the Rluc8. (F) Western Blot after deletion of the splice site shows full length expression of the biosensors and correction factor (Rluc8 alone). No splice variants could be detected. The box shows the base sequence of the splice site with one letter code for the corresponding amino acids above. In red marked the change of the thymine base into an adenine base to delete the splice site. The amino acid sequence did not change.
Fig 3
Fig 3. Characterization of eBRET2 biosensor expression after the deletion of the splice site.
(A) Luciferase activity via Luminescence measurement. Rluc8 expression correlates with Luciferase activity. (B) Western Blot shows full length expression of the biosensors, correction factor (Rluc8 alone) and maximum control Rluc8 GFP2. 1. Antibody: anti Rluc8 (1:1000) on the top and anti GAPDH (1:1000) on the bottom. 2. Antibody: anti Rabbit HRP (1:12000). (C) eBRET2 ratios of the biosensor variants normalized with the correction factor (Rluc8 30.1 ± 0.1 mBU). Significance defined as p < 0.001. (D) Delta eBRET2 ratios after incubation with 10 ng VEGF. Change in ΔBR of the maximum control (Rluc8 GFP2) was set as detection limit, marked with the line.
Fig 4
Fig 4
VEGF dependent Delta eBRET2 Ratios of the four biosensors Re02 (A), Re06 (B), Re07 (C) and Re09 (D). Biosensor concentrations in cell lysates were adjusted on the basis of RLuc8 expression to 200,000 RLU and 400,000 RLU. Lysates were incubated with individually adjusted VEGF concentrations and Delta eBRET2 Ratios were calculated. Significance defined as p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Bourne RRA, Flaxman SR, Braithwaite T, Cicinelli MV, Das A, Jonas JB et al. Magnitude, temporal trends, and projections of the global prevalence of blindness and distance and near vision impairment: a systematic review and meta-analysis. The Lancet Global Health 2017; 5(9):e888–e897. 10.1016/S2214-109X(17)30293-0 - DOI - PubMed
    1. Patil AS, Sable RB, Kothari RM. Occurrence, biochemical profile of vascular endothelial growth factor (VEGF) isoforms and their functions in endochondral ossification. J Cell Physiol 2012; 227(4):1298–308. 10.1002/jcp.22846 - DOI - PubMed
    1. Fuh G, Li B, Crowley C, Cunningham B, Wells JA. Requirements for Binding and Signaling of the Kinase Domain Receptor for Vascular Endothelial Growth Factor. The Journal of Biological Chemistry 1998; (Vol. 273, No. 18):11197–204. 10.1074/jbc.273.18.11197 - DOI - PubMed
    1. Holmes K, Roberts OL, Thomas AM, Cross MJ. Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal 2007; 19(10):2003–12. 10.1016/j.cellsig.2007.05.013 - DOI - PubMed
    1. Leppänen V-M, Prota AE, Jeltsch M, Anisimov A, Kalkkinen N, Strandin T et al. Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc Natl Acad Sci U S A 2010; 107(6):2425–30. 10.1073/pnas.0914318107 - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This study was funded in part by a graduate scholarship from the Justus-Liebig-University Giessen awarded to CS. No additional external funding was received for this study.