title: Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes.
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.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fridy
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Thompson
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Natalia E
authors.initials: NE
authors.lastname: Ketaren
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address: rout@rockefeller.edu.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D061905:Single-Domain Antibodies; D013205:Staphylococcal Protein A
keywords: Affinity isolation; Immunoprecipitation; Nanobody; Native elution; Protein A (PrA)