Applications of Sortase A from Staphylococcus aureus

Sortase A (SrtA) mediated ligation provides a mild and site-specific method for attaching a wide range of molecular probes to biological molecules, such as proteins and DNA. This method does not require any toxic reagents or harsh conditions to achieve high level of labelling, and the product could be easily isolated from the reaction mixture. This new labelling techniques could potentially improve exisiting technologies for studying molecular interations. A number of cloning and protein expression systems for the SrtA system were developed and the resulting proteins were very stable and in high purity. The use of this technique for site-specific protein labelling was investigated. A range of molecules, such as fluorecein derivitives and 25 bp double-stranded DNA, were successfully ligated to the target proteins in good yield. The use of the SrtA system in protein immobilisation was also thoroughly studied. A number of proteins with distintly different functionalties, such as fluorescent proteins (BFP, EGFP, DsRed), enzyme (Fpr) and DNA-binding protein (Tus), were successfully immobilised onto highly cross-linked polymeric beads (GMA), soft-polymer resin matrix (Affi-gel) and glass surfaces. The amount of non-specifically bound protein onto these surfaces was found to be neglectable and the activity of the attached protein was retained.EThOS - Electronic Theses Online ServiceGBUnited Kingdo