Engineering topics

Bradbury, Andrew M.

Publications and source records attributed to Bradbury, Andrew M..

2 records

DOE OSTI2021

Engineered pH-Sensitive Protein G/IgG Interaction

While natural protein–protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. Overall, we find that the new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Designed proteins for pH switchable antibody purification

Modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, a modified Fc-binding domain includes or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 6, or 8. Nucleic acids encoding the modified Fc-binding domains or polypeptides including one or more of the modified Fc-binding domains are also provided. In some embodiments, the nucleic acids are included in a vector and may be operably linked to a promoter. Methods for purifying a polypeptide including one or more Fc regions are also provided. Kits and reagents including the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are linked to a solid support.

Jha, Roshan Kumar↗