Sortase a engineering in escherichia coli using tripartite split green fluorescent protein system

Abstract

Staphylococcus aureus Sortase A, the trans-peptidase which ligate the protein to the gram-positive cell wall is widely used for the post-translational protein ligation. It has been highlighted due to its site-specific catalytic activity with short target motif. However Staphylococcus aureus sortase A is highly depends on the existence of $Ca^{2+}$ ion, therefore it is hard to utilize sortase A in vivo. To over-come this problem, I have developed high throughput screening system to isolate $Ca^{2+}$ independent SrtA mutant in Escherichia coli based on the tripartite split green fluorescent protein (GFP) system. Reassembly of tripartite split GFP catalyzed by $Ca^{2+}$ independent sortase A could be detected by high speed fluorescence activated cell sorter (FACS). After several round sorting, I found and characterized $Ca^{2+}$ independent sortase A mutant which had different mutation point with rationally engineered sort-ase A. The catalytic activity also validated using in vitro and in vivo analysis with several types of sub-strate. This research is not only an easy system of enzyme engineering, but also the first engineering application of tripartite split GFP system.