title: 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 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.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.affiliation: 1] Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. [2] Oncology Medicinal Chemistry, Janssen Research and Development, Beerse, Belgium.
authors.forename: Tongfei
authors.initials: T
authors.lastname: Wu
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimaraes
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Annet E M
authors.initials: AE
authors.lastname: Blom
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Ingram
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Zeyang
authors.initials: Z
authors.lastname: Li
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Lenka
authors.initials: L
authors.lastname: Kundrat
authors.affiliation: Centyrex, Janssen Research and Development, LLC, Spring House, Pennsylvania, USA.
authors.forename: Shalom D
authors.initials: SD
authors.lastname: Goldberg
authors.affiliation: 1] Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010458:Peptidyl Transferases / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D012685:Sepharose
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011506:Proteins; D012685:Sepharose; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases