Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Abstract

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879