The Expression,Purification And Polyclonal Antibody Preparation Of The TaSSP1, A Subtilisin-Like Protease In Wheat Leaves

Our laboratory has been working for many years on identifying endopeptidase isoenzymes in wheat senescencing leaves, a quite stable endopeptidase isoenzyme in wheat leaves has been detected recently. Then the protease was purified, its biochemical characteristics and primary structure were also studied. The result of MS (mass spectrometry) identification showed that the protease is a Subtilisin (subtilase)-like protease named as TaSSPl (Triticum aestivum Subtilisin-like Serine Protease1). The protease gene sequence verifed in GenBank by combining bioinformatics method. In order to understand better the physiological and biochemical characteristics of the protease and its localization in cell, the preparation of its polyclonal antibody is required, then the polyclonal antibody can be used to study science questions above and analyse the. mechanism of action in senescencing leaves of wheat, study then determinateSince the quantity of the protease in wheat leaves is quite low, it is difficult to directly purify it from the wheat leaves, in order to prepare the protease antibody, a recombinant protein, which possesses good antigen trait, can be expressed from E. coli instead of this nuture protease, firstly, according to NCBI mRNA sequence information Dong qiang cloned its718bp gene sequences fragment, TaSSP1-f (Triticum aestivum Subtilisin-like Serine Protease1-fragment), from wheat duck-reduced senescing leaves through RT-PCR, and then constructed the expression vector pET30a-TaSSP1-f On that basis, transforming recombinant plasmid into Rosetta (DE3) for IPTG-induced expression and the products of TaSSP1-f expression was analyzed by SDS-PAGE The result of experiments above proved that the gene sequences inserted into expression vector and the correct size of the fusion protein (TaSSP1-f) was expressed mainly in the form of inclusion bodies. The best inducing conditions for products:37℃,1.0mM IPTG, OD600=0.5and4h. The expressed inclusion bodies were washed with washing buffer before dissolving in8M Urea. The objective proteins were recovered from gel after SDS-PAGE and dissolved by PBS until its concentration was coming to2mg/mL, and then immunized two New Zealand rabbits by mixting with CFI or IFA according to1:1. Two and half months later the valence of polyclonal antibody in the rabbit’s antiserum was up to1:4. The results of Western-blot showed that this polyclonal antibody could not only bind the objective protein, but also specially bind the protease of wheat leaves in the gel separated by SDS-PAGE, the further research indicates that the quantity and activity of TaSSP1in the dark-reduced senescence wheat leaves regularly increases by time. So the polyclonal antibodies can be used in the follow-up study.