Design of protease-resistant peptide ligands for the purification of antibodies from human plasma
| Autor: | Amith D. Naik, Benjamin G. Bobay, Kevin Lawrence Ward, William S. Kish, Tuhidul Islam, Stefano Menegatti, Ruben G. Carbonell |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
medicine.medical_treatment Peptide Ligands 01 natural sciences Biochemistry Antibodies Chromatography Affinity Analytical Chemistry 03 medical and health sciences medicine Animals Chymotrypsin Humans Trypsin Binding site chemistry.chemical_classification Binding Sites Protease Chromatography 010401 analytical chemistry Organic Chemistry Proteolytic enzymes General Medicine 0104 chemical sciences Amino acid 030104 developmental biology chemistry Docking (molecular) IgG binding Peptides Blood Chemical Analysis Protein Binding medicine.drug |
| Zdroj: | Journal of Chromatography A. 1445:93-104 |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/j.chroma.2016.03.087 |
| Popis: | A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect