Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step
Abstract
:1. Introduction
2. Results and Discussion
2.1. The Pipeline
2.2. Affinity Selection and Mutagenesis
Targets | Full Names | Uniprot ID | Biological Processes | Antigens (Amino Acids #) |
---|---|---|---|---|
CDC34 | ubiquitin-conjugating enzyme E2 R1 | P49427 | ubiquitin ligase activity | 7–184 |
CDK2 | cyclin-dependent kinase 2 | P24941 | cell-cycle control | 3–286 |
COPS5 | COP9 signalosome complex subunit 5 | Q92905 | deubiquitination, JNK signaling, secretion | 9–309 |
CTBP1 | c-terminal-binding protein 1 | Q13363 | corepressor of transcriptional regulators | 20–440 |
MAP2K5 | mitogen-activated protein kinase kinase 5 | Q13163 | scaffold for the formation of a signaling process | 5–108 |
PAK1 | p-21 protein activated kinase 1 | Q13153 | regulation of cell-proliferation, apoptosis | 258–544 |
PLAA | phospholipase A-2-activating protein | Q9Y263 | maintenance of ubiquitin levels | 338–795 |
RAB6B | Ras-related protein-6B | Q9NRW1 | retrograde membrane trafficking via Golgi | 6–182 |
SF3A1 | Splicing factor 3A subunit 1 | Q15459 | mRNA processing, mRNA splicing | 423–790 |
TDP43 | TAR DNA-binding protein 43 | Q13148 | regulation of transcription and splicing | 1–106 |
USP11 | Ubiquitin carboxyl-terminal hydrolase 11 | P51784 | deubiquitination, regulator of NF-kappa-B activation | 61–285 |
2.3. Characterization of Affinity Matured Monobodies
Targets | Clones | BC Loop (26–30) | FG Loop (77–81) | Framework Mutations | Affinity (nM) | |
---|---|---|---|---|---|---|
ELISA | ITC | |||||
CDC34 | 1D10 | CGLWC | PGLRL | S17I, T76I | <50 | N/D |
COPS5 | 1D7 | RRWDV | WGIII | None | <10 | N/D |
MAP2K5 | 1C4 | CRKCL | RLEWL | P51H, K83N | 6 | 11 |
2C12 | CRKCL | RLEFL | None | 17 | 6 | |
SF3A1 | 1E2 | ALPVY | VWWYE | None | <50 | N/D |
USP11 | 1C2 | WWVPQ | PGIYQ | L18M, G61C, G65D, S82I | N/D | 4 |
1A9 | WWSVP | PGIYA | D67V, S82I, Y92C | 52 | N/D |
2.4. Pull-down Experiments with a Spiked HeLa Cell Lysate
3. Experimental Section
3.1. Subcloning, Overexpression and Purification of Antigens and Monobodies
3.2. Affinity Selection of the Primary Library
3.3. Secondary Library Construction and Affinity Selection
3.4. Competition ELISA to Estimate Binding Strength
3.5. Isothermal Titration Calorimetry (ITC)
3.6. Pull-down Assays
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Huang, R.; Gorman, K.T.; Vinci, C.R.; Dobrovetsky, E.; Gräslund, S.; Kay, B.K. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step. Int. J. Mol. Sci. 2015, 16, 23587-23603. https://doi.org/10.3390/ijms161023587
Huang R, Gorman KT, Vinci CR, Dobrovetsky E, Gräslund S, Kay BK. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step. International Journal of Molecular Sciences. 2015; 16(10):23587-23603. https://doi.org/10.3390/ijms161023587
Chicago/Turabian StyleHuang, Renhua, Kevin T. Gorman, Chris R. Vinci, Elena Dobrovetsky, Susanne Gräslund, and Brian K. Kay. 2015. "Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step" International Journal of Molecular Sciences 16, no. 10: 23587-23603. https://doi.org/10.3390/ijms161023587