| Study on the molecular mechanisms and regulatory processes of smooth muscle contractility is an important field for muscle research. The traditional MLCK pathway is still to be considered as a classic model, although many researches indicate the importance of the thin-filament binding proteins in smooth muscle contraction. Calponin(CaP) is a member of thin-filament binding proteins family. CaP can bind to F-actin and inhibit smooth muscle contractility, but the mechanism of CaP's effect on smooth muscle contraction is not clear yet.In this study, we found that the 2Ig-like module in the NH2-terminus of L-MLCK could bind to calponin. To further confirm it, we constructed the recombinant plasmids containing 2Ig-like modules of L-MLCK and transformed it into bacteria. The recombinant proteins 2Ig tagged with HA were expressed and purified. Then, we purified calponin from myofilaments by using ion-exchange chromatography. Finally, we used immunoprecipitation and overlap assay and confirmed the binding of the two proteins. This phenomenon indicates a new pathway of CaP involved in smooth muscle contraction and makes for the future work about its mechanism.Except for the above work , we also did some work about endostatin in this study. The bioactivity, refolding and multimer formation of endostatin, particularly of recombinant endostatin produced form bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, firstly, we expressed endostatin in E.Coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, then, by use of RP-HPLC, we successfully separated endostatin monomer and multimer for subjecting to...
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