Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Production of BAP-Tagged SH3 Domains
2.2. Phage Display Biopanning and Phage ELISAs
2.3. Affimer Protein Production
2.4. Production of GST-Tagged Proteins
2.5. Pull-Down of Affimer Proteins with GST-Tagged Full-Length Grb2
2.6. Pull-Down of Endogenous Grb2 with 6xHis-Tagged Affimer Proteins
2.7. SDS-PAGE and Western Blotting Analysis of Pull-Down Assays
2.8. Sulfhydryl Biotinylation of Affimer Protein
2.9. ELISA for Testing Affimer Cross-Reactivity
2.10. Production of Full-Length Recombinant Grb2
2.11. Fluorescence Polarisation (FP) Competition Assay
2.12. Mammalian Cell Culture
2.13. Mammalian Cell Transfection
2.14. Measuring Nuclear Phospho-ERK (p-ERK) in Transfected Cells
2.15. Generating U-2 OS Cells Stably Expressing tGFP-Tagged Affimer
2.16. Epidermal Growth Factor Receptor (EGFR) Signalling Assays in U-2 OS Cells Stably Expressing tGFP-Tagged Affimer
3. Results
3.1. Isolation of Affimer Proteins That Bind to the SH3 Domains of Grb2
3.2. Affimer Proteins Bound Full-Length Recombinant and Endogenous Grb2 in Pull-Down Assays
3.3. Affimer Proteins Bound Exclusively to Their Intended Target in an ELISA-Based Cross-Reactivity Assay
3.4. Affimer Proteins Isolated against Grb2 NSH3 Can Inhibit the Interaction between a SOS-Derived Peptide and Grb2
3.5. Affimer Protein Targeting the Grb2 CSH3 Alone Appears Sufficient for Curtailing Ras Signalling in Epidermal-Growth-Factor-Stimulated HEK-293 and U-2 OS Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Affimer Name | VR1 Sequence | VR2 Sequence | Frequency | Matching Clones |
---|---|---|---|---|
N-D7 | PIMVGEWNV | HRWFHKRFQ | 10/48 (20.8%) | N-B8, N-C8, N-H8, N-B9, N-E9, N-A10, N-C10, N-D10, N-E11 |
N-F10 | SVHKTDRFL | WKMIVTPPP | 9/48 (18.8%) | N-G7, N-A8, N-F8, N-H10, N-C11, N-D11, N-H11, N-H12 |
N-A9 | WYPSIIKPY | QTQFNNIPP | 7/48 (14.6%) | N-F7, N-H7, N-G11, N-C12, N-F12, N-G12 |
N-F11 | HRVGKHIMI | FNFSEPQQP | 2/48 (4.2%) | N-C7 |
N-G9 | IHVPPFDTW | YHRGSSWRI | 2/48 (4.2%) | N-F9 |
N-A12 | SDMYPKLHA | HISYGIAAN | 1/48 (2.1%) | |
N-C9 | SFVSKWKPY | DMPMSTKLK | 1/48 (2.1%) | |
N-B10 | WAHHMEPVA | FMGEGVFWI | 1/48 (2.1%) | |
N-G10 | WVQWQFEDM | YPGDMAYVA | 1/48 (2.1%) | |
N-D9 | HTFGRNVTE | FEWMWNPAN | 1/48 (2.1%) | |
N-H9 | HPYQVPVPA | HRRPKYIVP | 1/48 (2.1%) | |
N-B12 | KWDDLAWWP | ADFMLWEFV | 1/48 (2.1%) | |
N-A11 | NPYSPTVSG | YIYPKPTKY | 1/48 (2.1%) | |
N-E10 | PEQQAYQYN | YFAPSTWRI | 1/48 (2.1%) | |
N-A7 | EPFVPRTAWWW | QERQNHNMM | 1/48 (2.1%) | |
N-E12 | DGSPEKQI | GGRPWFIGR | 2/48 (4.2%) | N-E7 |
N-B11 | MIPHVE | AVWQRINFE | 2/48 (4.2%) | N-E8 |
C-D11 | IMRPYWASS | VGGDAYEKM | 1/48 (2.1%) | |
C-A8 | VMRPWWDSS | AAE | 16/48 (33.3%) | C-E7, C-F7, C-D8, C-G8, C-A9, C-B9, C-E9, C-F9, C-G9, C-H10, C-B11, C-C11, C-E11, C-H11, C-B12 |
C-D9 | IMRPWWHSS | AAE | 1/48 (2.1%) | |
C-F11 | IMRPWYMSS | AAE | 1/48 (2.1%) | |
C-C10 | IQRPWFYSS | AAE | 1/48 (2.1%) | |
C-B10 | IQRPFYDSS | AAE | 2/48 (4.2%) | C-C9 |
C-A11 | IKRPWWASS | AAE | 1/48 (2.1%) | |
C-C12 | RNIPIYPPQ | FEKPTSMHH | 3/48 (6.3%) | C-C7, C-E10 |
C-G11 | GPWGSVYMW | RETGNPTWL | 5/48 (10.4%) | C-B7, C-H8, C-A10, C-F12 |
C-F8 | DPYVIEVKD | AILGLIQPQ | 1/48 (2.1%) | |
C-C8 | AVQHWYPVQ | HGGHYRAPM | 1/48 (2.1%) | |
C-D10 | GNEIMHYNV | SWPTYWEPN | 1/48 (2.1%) |
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Tang, A.A.S.; Macdonald, A.; McPherson, M.J.; Tomlinson, D.C. Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation. Biomolecules 2024, 14, 1040. https://doi.org/10.3390/biom14081040
Tang AAS, Macdonald A, McPherson MJ, Tomlinson DC. Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation. Biomolecules. 2024; 14(8):1040. https://doi.org/10.3390/biom14081040
Chicago/Turabian StyleTang, Anna A. S., Andrew Macdonald, Michael J. McPherson, and Darren C. Tomlinson. 2024. "Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation" Biomolecules 14, no. 8: 1040. https://doi.org/10.3390/biom14081040