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

• Published in: Nature protocols Vol. 10; no. 3; pp. 508 - 516
• Main Authors: Witte, Martin D, Wu, Tongfei, Guimaraes, Carla P, Theile, Christopher S, Blom, Annet E M, Ingram, Jessica R, Li, Zeyang, Kundrat, Lenka, Goldberg, Shalom D, Ploegh, Hidde L
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.03.2015; Nature Publishing Group
• Subjects: 631/1647/666/2259; 631/92/607/1162; 631/92/612; 639/638/549/978; Aminoacyltransferases - genetics; Aminoacyltransferases - metabolism; Analytical Chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological Techniques; Catalysis; Chemistry; Chromatography; Computational Biology/Bioinformatics; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Enzymes; Enzymes, Immobilized - metabolism; Flow system; Labeling; Life Sciences; Lipids; Low temperature; Membrane proteins; Methods; Microarrays; Mutation - genetics; Organic Chemistry; Peptidyl Transferases - metabolism; Protein engineering; Protein Engineering - methods; Proteins; Proteins - metabolism; Protocol; R&D; Research & development; Sepharose; United States > US; Massachusetts
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2015.026

Sortase A can be used for the site-specific modification of proteins. This protocol describes its immobilization on Sepharose beads, allowing for larger-scale continuous flow reactions or easy separation from the enzyme in batch reactions. 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 Ca 2+ -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.