A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

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DOIResolve DOI: http://doi.org/10.1371/journal.pone.0163113
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TypeArticle
Journal titlePLOS ONE
ISSN1932-6203
Volume11
Issue9
Article numbere0163113
AbstractStaphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.
Publication date
PublisherPublic Library of Science
LanguageEnglish
AffiliationHuman Health Therapeutics; National Research Council Canada
Peer reviewedYes
NRC numberNRC-HHT-53324
NPARC number23000783
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Record identifierf43c114f-2618-4773-9929-bcb2d6fee7ba
Record created2016-09-22
Record modified2017-05-09
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