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A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies
Staphylococcal 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. Curr...
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| Published in: | PloS one 2016-09, Vol.11 (9), p.e0163113 |
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| creator | Henry, Kevin A Sulea, Traian van Faassen, Henk Hussack, Greg Purisima, Enrico O MacKenzie, C Roger Arbabi-Ghahroudi, Mehdi |
| description | Staphylococcal 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. |
| doi_str_mv | 10.1371/journal.pone.0163113 |
| format | article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0163113</identifier><identifier>PMID: 27631624</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Affinity ; Affinity chromatography ; Animals ; Antigens ; Binding ; Bioinformatics ; Biology and Life Sciences ; Camelids, New World - immunology ; Cancer ; Chromatography ; Councils ; Environmental science ; Frequency variation ; Genes ; Health aspects ; Immunoglobulins ; Immunological tolerance ; Medicine and Health Sciences ; Monoclonal antibodies ; Mutagenesis ; Mutation ; Nanobodies ; Physical Sciences ; Protein A ; Protein binding ; Protein Engineering ; Protein G ; Proteins ; Purification ; Research and analysis methods ; Single-Domain Antibodies - genetics ; Staphylococcal Protein A - genetics ; Streptococcal protein G ; Streptococcus ; Therapeutic applications</subject><ispartof>PloS one, 2016-09, Vol.11 (9), p.e0163113</ispartof><rights>COPYRIGHT 2016 Public Library of Science</rights><rights>2016 Henry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2016 Henry et al 2016 Henry et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c725t-f92035d6a33998c79fb517938a02b723ffa396976c08f0b85e478da3469abfe63</citedby><cites>FETCH-LOGICAL-c725t-f92035d6a33998c79fb517938a02b723ffa396976c08f0b85e478da3469abfe63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/1819912012/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/1819912012?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,315,730,783,787,888,25765,27936,27937,37024,37025,44602,53804,53806,75460</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27631624$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Goldman, Ellen R</contributor><creatorcontrib>Henry, Kevin A</creatorcontrib><creatorcontrib>Sulea, Traian</creatorcontrib><creatorcontrib>van Faassen, Henk</creatorcontrib><creatorcontrib>Hussack, Greg</creatorcontrib><creatorcontrib>Purisima, Enrico O</creatorcontrib><creatorcontrib>MacKenzie, C Roger</creatorcontrib><creatorcontrib>Arbabi-Ghahroudi, Mehdi</creatorcontrib><title>A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Staphylococcal 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.</description><subject>Affinity</subject><subject>Affinity chromatography</subject><subject>Animals</subject><subject>Antigens</subject><subject>Binding</subject><subject>Bioinformatics</subject><subject>Biology and Life Sciences</subject><subject>Camelids, New World - immunology</subject><subject>Cancer</subject><subject>Chromatography</subject><subject>Councils</subject><subject>Environmental science</subject><subject>Frequency variation</subject><subject>Genes</subject><subject>Health aspects</subject><subject>Immunoglobulins</subject><subject>Immunological tolerance</subject><subject>Medicine and Health Sciences</subject><subject>Monoclonal antibodies</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Nanobodies</subject><subject>Physical Sciences</subject><subject>Protein A</subject><subject>Protein binding</subject><subject>Protein Engineering</subject><subject>Protein G</subject><subject>Proteins</subject><subject>Purification</subject><subject>Research and analysis methods</subject><subject>Single-Domain Antibodies - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27631624</pmid><doi>10.1371/journal.pone.0163113</doi><tpages>e0163113</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| source | PubMed Central (Open access); Publicly Available Content Database |
| subjects | Affinity Affinity chromatography Animals Antigens Binding Bioinformatics Biology and Life Sciences Camelids, New World - immunology Cancer Chromatography Councils Environmental science Frequency variation Genes Health aspects Immunoglobulins Immunological tolerance Medicine and Health Sciences Monoclonal antibodies Mutagenesis Mutation Nanobodies Physical Sciences Protein A Protein binding Protein Engineering Protein G Proteins Purification Research and analysis methods Single-Domain Antibodies - genetics Staphylococcal Protein A - genetics Streptococcal protein G Streptococcus Therapeutic applications |
| title | A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies |
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