summary
Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS–PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wilchek, M. & Bayer, E. A. (1999) Foreword and introduction to the book (strept)avidin-biotin system, Biomol. Eng. 16, 1–4.
Hamblett, K. J., Kegley, B. B., Hamlin, D. K., Chyan, M. K., Hyre, D. E., Press, O. W., Wilbur, D. S. & Stayton, P. S. (2002) A streptavidin-biotin binding system that minimizes blocking by endogenous biotin, Bioconjug. Chem. 13, 588–98.
Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Stayton, P. S., To, R., Klumb, L. A., Buhler, K. R. & Vessella, R. L. (1999) Development of new biotin/streptavidin reagents for pretargeting, Biomol. Eng. 16, 113–8.
van Osdol, W. W., Sung, C., Dedrick, R. L. & Weinstein, J. N. (1993) A distributed pharmacokinetic model of two-step imaging and treatment protocols: application to streptavidin-conjugated monoclonal antibodies and radiolabeled biotin, J. Nucl. Med. 34, 1552–64.
Nakamura, M., Tsumoto, K., Ishimura, K. & Kumagai, I. (2002) Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate, Anal. Biochem. 304, 231–5.
Lee, B. S., Gupta, S., Krishnanchettiar, S. & Lateef, S. S. (2005) Capturing SDS-treated biotinylated protein and peptide by avidin functional affinity electrophoresis with or without SDS in the gel running buffer, Anal. Biochem. 336, 312–5.
Fukushima, H., Morgan, H. & Taylor, D. M. (1994) Self-assembly of avidin and streptavidin with multifunctional biotin molecules, Thin Solid Films 244, 789–93.
Diamandis, E. P. & Christopoulos, T. K. (1991) The biotin-(strept)avidin system: principles and applications in biotechnology, Clin. Chem. 37, 625–36.
Collot, J., Gradinaru, J., Humbert, N., Skander, M., Zocchi, A. & Ward, T. R. (2003) Artificial metalloenzymes for enantioselective catalysis based on biotin-avidin, J. Am. Chem. Soc. 125, 9030–1.
Skander, M., Humbert, N., Collot, J., Gradinaru, J., Klein, G., Loosli, A., Sauser, J., Zocchi, A., Gilardoni, F. & Ward, T. R. (2004) Artificial metalloenzymes: (strept)avidin as host for enantioselective hydrogenation by achiral biotinylated rhodium-diphosphine complexes, J. Am. Chem. Soc. 126, 14411–8.
Letondor, C., Humbert, N. & Ward, T. R. (2005) Artificial metalloenzymes based on the biotin-avidin technology for the enantioselective reduction of ketones via transfer-hydrogenation, Proc Natl Acad Sci USA 102, 4683–7.
Zocchi, A., Humbert, N., Berta, T. & Ward, T. R. (2003) Efficient expression and mutation of avidin and streptavidin as host proteins for enantioselective catalysis, Chimia 57, 589–92.
Reetz, M. T. (2004) Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications, Proc Natl Acad Sci USA 101, 5716–22.
Reetz, M. T. (2001) Combinatorial and evolution-based methods in the creation of enantioselective catalysts, Angew. Chem. Int. Ed. Engl. 40, 284–310.
Kada, G., Falk, H. & Gruber, H. J. (1999) Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate, Biochim. Biophys. Acta. 1427, 33–43.
Humbert, N., Zocchi, A. & Ward, T. R. (2005) Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for biotin-binding proteins, Electrophoresis 26, 47–52.
Sambrock, J. & Russel, D. W. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY, 2001.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680–5.
Green, N. M. (1975) Avidin, Adv. Protein Chem. 29, 85–133.
Freitag, S., Le Trong, I., Chilkoti, A., Klumb, L. A., Stayton, P. S. & Stenkamp, R. E. (1998) Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex, J. Mol. Biol. 279, 211–21.
Acknowledgments
We thank Prof. C. R. Cantor for the streptavidin gene as well as Profs. P. Schürmann and J.-M. Neuhaus for their help in setting up the protein production. This work was funded by the Swiss National Science Foundation (Grants FN 620-57866.99 and FN 200021-105192/1 as well as NRP 47 “Supramolecular Functional Materials”), CERC3 (Grant FN20C321-101071), the Roche Foundation, the Canton of Neuchâtel as well as FP6 Marie Curie Research Training Network (IBAAC network, MRTN-CT-2003-505020).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Humbert, N., Ward, T.R. (2008). Functionality Screen of Streptavidin Mutants by Non-Denaturing SDS–PAGE Using Biotin-4-Fluorescein. In: McMahon, R.J. (eds) Avidin-Biotin Interactions. Methods In Molecular Biology™, vol 418. Humana Press. https://doi.org/10.1007/978-1-59745-579-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-59745-579-4_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-583-5
Online ISBN: 978-1-59745-579-4
eBook Packages: Springer Protocols