title: Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus.
abstract: Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps.
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Brüderstr. 34, 04103 Leipzig, Germany. msteinha@uni-leipzig.de
authors.forename: Max
authors.initials: M
authors.lastname: Steinhagen
authors.forename: Katja
authors.initials: K
authors.lastname: Zunker
authors.forename: Karoline
authors.initials: K
authors.lastname: Nordsieck
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016209:Interleukin-8 / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D016209:Interleukin-8; D010455:Peptides; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases