Abstract
The Strep-tag is a nine-residue minimal peptide sequence with intrinsic affinity towards streptavidin. This peptide – or its improved version Strep-tag II – can be fused to recombinant antibody fragments and other proteins in various fashions. The fact that the interaction between the Strep-tag and streptavidin is highly specific and can be reversed upon addition of biotin (or suitable derivatives such as desthiobiotin) enables a highly efficient affinity chromatography under biochemically mild conditions. Here we describe a protocol for the production and purification of Strep-tagged antibody fragments from bacterial cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), followed by analysis in ELISA and on Western blots using Strep-Tactin enzyme conjugates or anti-Strep-tag antibodies. Thus, the Strep-tag, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of antibody fragments and for their detection or molecular analysis.
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Schlapschy, M., Skerra, A. (2010). Purification and Analysis of Strep-tagged Antibody-Fragments. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01147-4_25
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DOI: https://doi.org/10.1007/978-3-642-01147-4_25
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01146-7
Online ISBN: 978-3-642-01147-4
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