Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling
| Autor: | Julie Nigro, Mariusz P. Madej, Samuel Baer, Gregory Coia, Stewart D. Nuttall, Randy Suryadinata, Rebecca M. Nisbet, Timothy E. Adams, Lisa P. T. Hong, Charlotte C. Williams |
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| Rok vydání: | 2014 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Staphylococcus aureus Molecular Sequence Data Lysine Fluorescent Antibody Technique Peptide Protein Engineering Biochemistry Immunoglobulin Fab Fragments Mice Bacterial Proteins Nuclear Matrix-Associated Proteins Animals Amino Acid Sequence Physical and Theoretical Chemistry Peptide sequence Cells Cultured Neurons chemistry.chemical_classification Amyloid beta-Peptides Bioconjugation Organic Chemistry Protein engineering Aminoacyltransferases Embryo Mammalian Ethylenediamines Fusion protein Recombinant Proteins Amino acid Cysteine Endopeptidases chemistry Sortase A Oligopeptides Single-Chain Antibodies |
| Zdroj: | Org. Biomol. Chem.. 12:2675-2685 |
| ISSN: | 1477-0539 1477-0520 |
| DOI: | 10.1039/c3ob42325e |
| Popis: | The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal. |
| Databáze: | OpenAIRE |
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