Affinity extraction of proteins from acidic environments (979.2)

Abstract

Affinity extraction of proteins from acidic environments Affinity purification is widely used to isolate an analyte of interest from a complex mixture, such as a cell lysate. The process involves immobilization of one ligand followed by incubation with a sample containing the binding partner. While most affinity purifications are performed at near‐physiological pH, there are situations where performing affinity extraction at low pH would be advantageous, including isolating proteins from acidic environments such as the stomach or acidic hot springs. The affinity pairs most often used for protein purification (e.g., antibody:antigen, GST‐tags:glutathione or immobilized metals:6xHis tags) have limited or abolished binding at low pH. Here we employed a published double mutant of streptavidin which is known to form a tight complex with biotin at low pH. A vector designed to incorporate an N‐terminal biotinylation sequence onto a protein was used to direct in vivo biotinylation of a selected lysine residue in several target proteins. This strategy has advantages over non‐specific in vitro, post‐purification biotinylation wherein multiple labels are added randomly and thereby cause disruption of key protein‐protein interactions. We demonstrate the utility of the mutant streptavidin:biotin system for extracting proteins of interest by characterizing the binding kinetics of this affinity pair in various solutions, pH’s and temperatures. Support: NIH R01‐GM101135Grant Funding Source: Supported by NIH R01‐GM101135