Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin

Müller J, Risse JM, Friehs K, Flaschel E (2015)
Engineering in Life Sciences 15(6): 627-639.

Journal Article | Published | English

No fulltext has been uploaded

Abstract
The protein streptavidin (SAV) is applied in a large variety of molecular methods due to an extraordinarily strong binding to the vitamin biotin (BIO). The protein structure is homotetrameric, characterized by one binding site for BIO per subunit. Therefore, one of the major criteria to determine the quality of SAV isolates is the proportion of BIO-blocked binding sites per tetramer. A rapid analysis of BIO-free binding sites is achieved by fluorescence quenching of biotin-4-fluorescein (B4F). However, BIO-blocked binding sites can only be determined by costly and laborious procedures such as ELISA-based methods or radioactive labeling. This study describes the systematic, model-supported development of a method for the quick and simple detection of BIO-blocked binding sites, based on a short-term heat incubation of the sample in the presence of B4F. Kinetic modeling and parameter estimation yielded dissociation constants of 1.22 +/- 0.27 x 10(-11) M for the complex SAV-BIO and 5.16 +/- 0.70 x 10(-13) M for SAV-B4F at 70 degrees C, allowing a displacement of SAV-bound BIO by B4F. This method allows the rapid monitoring of BIO-blocked binding sites in fermentation processes, independent from the chain length of SAV and the concentration of contaminating proteins, e.g. when optimizing the BIO concentration in cultivation media.
Publishing Year
ISSN
PUB-ID

Cite this

Müller J, Risse JM, Friehs K, Flaschel E. Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin. Engineering in Life Sciences. 2015;15(6):627-639.
Müller, J., Risse, J. M., Friehs, K., & Flaschel, E. (2015). Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin. Engineering in Life Sciences, 15(6), 627-639.
Müller, J., Risse, J. M., Friehs, K., and Flaschel, E. (2015). Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin. Engineering in Life Sciences 15, 627-639.
Müller, J., et al., 2015. Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin. Engineering in Life Sciences, 15(6), p 627-639.
J. Müller, et al., “Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin”, Engineering in Life Sciences, vol. 15, 2015, pp. 627-639.
Müller, J., Risse, J.M., Friehs, K., Flaschel, E.: Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin. Engineering in Life Sciences. 15, 627-639 (2015).
Müller, Jakob, Risse, Joe Max, Friehs, Karl, and Flaschel, Erwin. “Model-based development of an assay for the rapid detection of biotin-blocked binding sites of streptavidin”. Engineering in Life Sciences 15.6 (2015): 627-639.
This data publication is cited in the following publications:
This publication cites the following data publications:

Export

0 Marked Publications

Open Data PUB

Web of Science

View record in Web of Science®

Search this title in

Google Scholar