Research On The Validity Of Spyligase System In Saccharomyces Cerevisiae

SpyLigase system is based on SpyTag/SpyCatcher interaction and reasonably reformed from it. It consists of KTag which is a peptide tag, SpyTag which is also a peptide, and SpyLigase which is a ligase, the three parts generate a covalent peptide-peptide ligation system. Both KTag and SpyTag can form a spontaneous isopeptide bonds with SpyLigase.SpyLigase sytstem could link proteins to build blocks with minimal modifications. This system Has important theoretical significance on the construction of multi-emzyme complex and supermolecule, and the conformation of proteins; it can be used to conform a variety of configurations of proreins, emzymes and sustained relase prorein drugs. At present, SpyLigase system was only single-conponent expressed in Escherichia coli, the interaction was preceed in vitro. It had never been verified in eucaryote cells.In this study, we used Saccharomyces cerevisiae, a kind of eucaryote, as host, verified the effectiveness of SpyLigase in eucaryote cells. Firstly, we constructed three plasmids,they were YEplac112-PSLT (containing the promoter induced by Cu2+ and SpyLigase gene),YEplac181-PT-HSTeG (containing the promoter induced by Cu2+, and green fluorescent protein gene with his×6 tag and SpyTag), and YEplacl95-PT-RKTHA (containing the promoter induced by Cu2+, and red fluorescent protein gene with KTag). Three constructed plasmids were transformed into S. cerevisiae, as objective strain. We also set one negetive control and three positive controls. At late logarithmic growth of the negative control strains, the positive control strain 1 and the positive control strain 2, respectively, Cu2+ was added. These strains were incubated for some time and then detected by fluorescence microscopy. It is verified that three empty vectors woldn't affect the experiment and the constructed plasmids could expressed in S. cerevisiae. At late logarithmic growth of the positive control strain 3 and the objective strain respectively, Cu2+ was added. These strains were incubated for some time. Then we extract the holoprotein of these strains and purified by his×6 tag, respectively. At last, the purified proteins were detected by fluorescence microscopy. After observing the green fluorescence and the red fluorescence of the purified proteins of the positive control strain 3 and the objective strain, we verified the effectiveness of SpyLigase system in S.cerevisiae.