title: Design of protease-resistant peptide ligands for the purification of antibodies from human plasma.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Campux Box 7928, Raleigh, NC 27695, USA.
authors.forename: Stefano
authors.initials: S
authors.lastname: Menegatti
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695, USA.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Bobay
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Ward
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA.
authors.forename: Tuhidul
authors.initials: T
authors.lastname: Islam
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Kish
authors.affiliation: Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Campux Box 7928, Raleigh, NC 27695, USA.
authors.forename: Amith D
authors.initials: AD
authors.lastname: Naik
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Campux Box 7928, Raleigh, NC 27695, USA. Electronic address: ruben@ncsu.edu.
authors.forename: Ruben G
authors.initials: RG
authors.lastname: Carbonell
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001665:Binding Sites; D001774:Blood Chemical Analysis / Q000379:methods*; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism
chemical_list: D000906:Antibodies; D008024:Ligands; D010455:Peptides; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014357:Trypsin
keywords: Affinity chromatography; Molecular docking; Peptide affinity ligands; Polyclonal antibodies; Protease; Proteolysis