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Anal Biochem. 2015 May 15;477:92-4. doi: 10.1016/j.ab.2015.02.013. Epub 2015 Feb 21.

Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes.

Author information

1
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA.
2
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address: rout@rockefeller.edu.

Abstract

In addition to its high affinity for antibody Fc domains, staphylococcal Protein A has been shown to bind certain Fab domains. We investigated this in order to develop a small, recombinant Protein A-binding alternative to immunoglobulin G (IgG) from nanobodies, single-domain antibodies derived from a camelid variant IgG's variable region. We engineered a nanobody with affinity solely for Protein A as well as a dimerized version of higher affinity for typical multidomain Protein A constructs. Because this recombinant nanobody can be immobilized using a cleavable crosslinker, it has proven to be suitable for the isolation and mild elution of protein complexes in native conditions.

KEYWORDS:

Affinity isolation; Immunoprecipitation; Nanobody; Native elution; Protein A (PrA)

PMID:
25707320
PMCID:
PMC4404190
DOI:
10.1016/j.ab.2015.02.013
[Indexed for MEDLINE]
Free PMC Article

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