| Sortase A transpeptidase, a membrane protein of Staphylococcus aureus, mediates specific attachment of various components such as enzymes, pilins, and glycoproteins to cell wall. This enzyme cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of cell-wall peptidoglycan. The high linking specificity and efficiency makes sortase a good resource for protein-nucleophilic molecules linking, such as protein-protein ligation, protein-necleic acid ligation, protein cyclization, live cell labeling, etc, therefore demonstrating great promise on its wide application in biological research and biopharmaceutical industry.In this study, wildtype sortase A (cloned from Staphylococcus aureus), and high activity mutant in high purity were prepared recombinantly from E. coli, enzyme production of per liter culture medium is 30 mg; also, model substrates were carefully designed and prepared to monitor the ligation reaction in a sensitive manner. Then, high efficiency ligation prodecure based on high activity Sortase A mediated protein and biotinylated peptide ligation is establishmentTaking advantages of this prodecure, a protein called SpyCatcher (SC) was able to be site-specifically labeled by a biotin group, purity is 95%, incubation of the biotinylated SC protein and streptavidin-coated magnetic beads resulted in highly oriented immobilization of recombinant protein on solid surface, binding capacity is O1μg/μL. It’s proved that the high efficiency ligation prodecure can be successfully appilied, and provided an example for the better and more widely used.Successful preparation of high activity sortase A mutant and establishment of high efficiency ligation procedure will not only facilitate highly oriented immobilization of proteins, but also lay a solid foundation for future application of this versatile tool in other biotechnology fields. |
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