Title
Protein engineering allows for mild affinity-based elution of therapeutic antibodies
Author
Sara Kanje, Raminta Venskutonytė, Julia Scheffel, Johan Nilvebrant, Karin Lindkvist-Petersson, Sophia Hober
Year
2018
Journal
Journal of Molecular Biology
Abstract
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid (EDTA). Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa-Fc-complex the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Ligand binding, Thermal stability, Thermodynamics, Biochemistry