Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region
Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still disp...
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| Veröffentlicht in: | Bioconjugate chemistry 2018-08, Vol.29 (8), p.2763-2775 |
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| container_title | Bioconjugate chemistry |
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| creator | Kruljec, Nika Molek, Peter Hodnik, Vesna Anderluh, Gregor Bratkovič, Tomaž |
| description | Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity (K D app. 0.6 μM) compared to the parent peptide. When coupled to paramagnetic beads or a chromatographic matrix, the optimized ligand was shown to selectively enrich antibodies from complex protein mixtures. |
| doi_str_mv | 10.1021/acs.bioconjchem.8b00395 |
| format | Article |
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However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity (K D app. 0.6 μM) compared to the parent peptide. 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However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity (K D app. 0.6 μM) compared to the parent peptide. When coupled to paramagnetic beads or a chromatographic matrix, the optimized ligand was shown to selectively enrich antibodies from complex protein mixtures.</description><subject>Affinity</subject><subject>Affinity chromatography</subject><subject>Alanine</subject><subject>Amino Acid Sequence</subject><subject>Antibodies</subject><subject>Antigen presentation</subject><subject>Bacteriophages - genetics</subject><subject>Beads</subject><subject>Binding</subject><subject>Chromatography, Affinity - methods</subject><subject>Combinatorial analysis</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Glutamine</subject><subject>Humans</subject><subject>Immunoglobulin Fc Fragments - chemistry</subject><subject>Immunoglobulin Fc Fragments - genetics</subject><subject>Immunoglobulin G</subject><subject>Immunoglobulin G - chemistry</subject><subject>Immunoglobulin G - genetics</subject><subject>Ligands</subject><subject>Molecular biology</subject><subject>Molecules</subject><subject>Mutagenesis</subject><subject>Peptides</subject><subject>Peptides - chemistry</subject><subject>Peptides - metabolism</subject><subject>Phages</subject><subject>Protein A</subject><subject>Proteins</subject><subject>Screens</subject><subject>Staphylococcal Protein A - metabolism</subject><subject>Surface Plasmon Resonance</subject><issn>1043-1802</issn><issn>1520-4812</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkF1LwzAUhoMobn78BS14403nSdKsyaVMncLAD_Q6pOnp7Gib2rSC_nozNkW8kVyccHjeN-Eh5JTChAKjF8b6SVY665qVfcV6IjMArsQOGVPBIE4kZbvhDgmPqQQ2IgferwBAUcn2yYgDsCTl6Zg8XuE7Vq6tsekj0-TR7NV0xvbYlZ-mL10TuSJ6wLYvc4wW5TIgfr26q-uhccvKZUNVNtE8urHREy5D4IjsFabyeLydh-Tl5vp5dhsv7ud3s8tFbLgUfWyzzORKTCXLkAtKhWWCWQE8n-YsUSmVhQ0HmZICVQK5AZlITilYUdgs54fkfNPbdu5tQN_ruvQWq8o06AavGaScU6X4NKBnf9CVG7om_E4zGpBEcFCBSjeU7Zz3HRa67cradB-agl5b18G6_mVdb62H5Mm2f8hqzH9y35oDwDfAuuHn7f9qvwALC5Mz</recordid><startdate>20180815</startdate><enddate>20180815</enddate><creator>Kruljec, Nika</creator><creator>Molek, Peter</creator><creator>Hodnik, Vesna</creator><creator>Anderluh, Gregor</creator><creator>Bratkovič, Tomaž</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-8367-5465</orcidid></search><sort><creationdate>20180815</creationdate><title>Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region</title><author>Kruljec, Nika ; Molek, Peter ; Hodnik, Vesna ; Anderluh, Gregor ; Bratkovič, Tomaž</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a385t-cbbad95682be35115c252c503d6d249718fcfcfe2985e940da08483110c5fcbd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Affinity</topic><topic>Affinity chromatography</topic><topic>Alanine</topic><topic>Amino Acid Sequence</topic><topic>Antibodies</topic><topic>Antigen presentation</topic><topic>Bacteriophages - genetics</topic><topic>Beads</topic><topic>Binding</topic><topic>Chromatography, Affinity - methods</topic><topic>Combinatorial analysis</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Glutamine</topic><topic>Humans</topic><topic>Immunoglobulin Fc Fragments - chemistry</topic><topic>Immunoglobulin Fc Fragments - genetics</topic><topic>Immunoglobulin G</topic><topic>Immunoglobulin G - chemistry</topic><topic>Immunoglobulin G - genetics</topic><topic>Ligands</topic><topic>Molecular biology</topic><topic>Molecules</topic><topic>Mutagenesis</topic><topic>Peptides</topic><topic>Peptides - chemistry</topic><topic>Peptides - metabolism</topic><topic>Phages</topic><topic>Protein A</topic><topic>Proteins</topic><topic>Screens</topic><topic>Staphylococcal Protein A - metabolism</topic><topic>Surface Plasmon Resonance</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kruljec, Nika</creatorcontrib><creatorcontrib>Molek, Peter</creatorcontrib><creatorcontrib>Hodnik, Vesna</creatorcontrib><creatorcontrib>Anderluh, Gregor</creatorcontrib><creatorcontrib>Bratkovič, Tomaž</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Bioconjugate chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kruljec, Nika</au><au>Molek, Peter</au><au>Hodnik, Vesna</au><au>Anderluh, Gregor</au><au>Bratkovič, Tomaž</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region</atitle><jtitle>Bioconjugate chemistry</jtitle><addtitle>Bioconjugate Chem</addtitle><date>2018-08-15</date><risdate>2018</risdate><volume>29</volume><issue>8</issue><spage>2763</spage><epage>2775</epage><pages>2763-2775</pages><issn>1043-1802</issn><eissn>1520-4812</eissn><abstract>Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity (K D app. 0.6 μM) compared to the parent peptide. 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| subjects | Affinity Affinity chromatography Alanine Amino Acid Sequence Antibodies Antigen presentation Bacteriophages - genetics Beads Binding Chromatography, Affinity - methods Combinatorial analysis Enzyme-Linked Immunosorbent Assay Glutamine Humans Immunoglobulin Fc Fragments - chemistry Immunoglobulin Fc Fragments - genetics Immunoglobulin G Immunoglobulin G - chemistry Immunoglobulin G - genetics Ligands Molecular biology Molecules Mutagenesis Peptides Peptides - chemistry Peptides - metabolism Phages Protein A Proteins Screens Staphylococcal Protein A - metabolism Surface Plasmon Resonance |
| title | Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region |
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