Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro

Li Ye1 Chen Lei1 Zhou Jun1 Li Chenghua1 Su Xiurong1 Li Taiwu2(1.School of Marine Sciences,Ningbo University,Ningbo 315211;2.Ningbo City College of Vocational Technology,Ningbo 315100)

Dendrorhynchus zhejiangensis Actin Prokaryotic expression Atomic force microscope Self-assembly

Actin is a major component of cytoskeleton.In cells,actin associates with actin binding proteins(ABPs)to form highly ordered polymerization structure on the basis of F-actin,which plays a variety of important physiological functions.According to the full length cDNA of Dendrorhynchus zhejiangensis actin,the prokaryotic expression plasmid for actin(pET-28a-A)was constructed and transformed into E.coliBL21(DE3).After IPTG induction,the recombinant proteins were highly expressed and contained in inclusion bodies.The recombinant proteins from inclusion bodies were purified by Nickel nitrilotriacetic(Ni-NTA)agarose affinity chromatography,and further dialyzed into a refolding buffer.The results of sodium dodecyl sulfate polycrylamide gel(SDS-PAGE)showed that the relative molecular weight of r-actin was 43 kD.In in vitro polymerization condition,the atomic force microscope(AFM)was used to observe and analyze the large-scale aggregate structure of r-actin during self-assembly.Our results showed that in vitro assembly of r-actin polymerized into complicated discrete structures,like filopodia-like fiber bundles,random coiled actin clusters,in addition to form disorganized protein aggregates.These data implicate that in vitro assembly of r-actin reflects its inherent thermodynamic properties of polymerization;further study would help to explore the regulatory function of actin binding proteins(ABPs)in the assembly of actin supramolecular aggregate structures.