Chaperone Activity Of Conservative GTPase

GTPase is a large group of conservative enzymes which plays a crucial role in cell.GTPase widely participates and plays an important biological function in signal transduction, protein synthesis and transport, cell differentiation and energy metabolism. From bacterial genome sequence analysis it is found that 11 GTPases in bacteria are highly conservative.These conservative GTPases are associated with ribosomes, helping ribosome to work. Ribosomes is the factory of protein synthesis. Peptide do not have biological activity when it is translated from ribosome. Peptide do not have stable 3d structure when it is translated from ribosome easily mutual together to form precipitation. Cells had various function molecular chaperone, formed very complicated network system in order to ensure the quality of the protein inside the body.Several papers report that EF-G, IF2, EF-Tu have the activity of chaperone.They can help protein folding.But there are few reports of other conservative GTPases. This paper studies the chaperone activity of conservative GTPase except LepA. In this paper we clone eight kinds of gene of E. coli GTPase into pET-28a and pET-22b vectors,then transform the vectors into E. coli (BL21) to purify proteins. Protein purification process of preliminary purification using Ni column, cationic exchange column (SP column), scanty water column. Futher purification using molecular sieve, desalination column. We succeed in purifying six GTPases but failed in EngA.Chaperone activeity of GTPase were tested by using renaturation of citrate synthase and a-glucosidase.As the experiment results shows, the conservative E. coli GTPases can promote the renaturation of citric acid synthase,but of different degree proposed from low to high. Six kinds of GTPases can help the renaturation of a-glucosidase except Ffh.In renaturation buffer Ffh and a-glucosidase form precipitation because of different electrical charge.E. coli GTPase has chaperone activity to a certain extant because they are conservative.They may help the nascent peptide folding before the apperance of special chaperone protein.Cells may rely mainly on these protein to help new peptide folding and maintain protein stability.From the result of the citrate synthase andα-glucosidase renaturation experiments we found that protein ObgE has a much higher chaperone activity.We use dynamic light scattering to test the uniformity of Protein ObgE solution.We find that ObgE protein has very good uniformity.99.4% mass scattering light of protein solution are from Radius 3.33 nm.We test ObgE chaperone activity by using renaturation bovine carbonic anhydraseⅡ. The result shows that ObgE can help the renaturation of BCAⅡ.The result will have impact on the research of these conservative GTPases.