Focused peptide library screening as a route to a superior affinity ligand for antibody purification
| Autor: | Barbara Jenko Bizjan, Krištof Bozovičar, Jernej Kovač, A. Meden, Tomaž Bratkovič |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Peptide udc:543.544+577.27 Ligands 01 natural sciences Biochemistry Chromatography Affinity biophysics chemistry.chemical_classification Multidisciplinary Downstream processing Sepharose peptidne knjižnice High-Throughput Nucleotide Sequencing Directed evolution Chemical biology imunoglobulini Medicine ELISA biotechnology Protein Binding Biotechnology Science Biophysics chemical biology Article 03 medical and health sciences Affinity chromatography Peptide Library biochemistry Humans Amino Acid Sequence Peptide library Staphylococcal Protein A Ligand 010401 analytical chemistry kromatografija Fragment crystallizable region 0104 chemical sciences High-Throughput Screening Assays Immunoglobulin Fc Fragments 030104 developmental biology chemistry Alcohols Immunoglobulin G analizna kemija Directed Molecular Evolution Linker |
| Zdroj: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-12 (2021) Scientific reports, vol. 11, no. 11650, 2021. |
| ISSN: | 2045-2322 |
| Popis: | Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin. |
| Databáze: | OpenAIRE |
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