Preliminary Functional Study Of In Vitro Evolution ATP-binding Protein

We proposed a hypothesis that the original protein structure is induced by small molecules through the analysis of the conservative property of small molecular ligands.We used the c DNA display technique to validate the hypothesis and achieved the most prevalent sequence. The selected peptide exhibits ATP affinity and ATPhydrolysis.On the basis of this study, we used experimental methods to detect the selected protein’s physical and chemical properties and structure.Then selected the original protein’s interaction protein in a c DNA library.First, using Nco I and Eco R I as enzyme cutting sites, we redesigned the specific primers to construct a prokaryotic expression vector: p ET-28b-abp1, which contain the most prevalent sequence.The protein’s concentration is 2.11 mg/m L.The protein’s secondary structure was analyzed by NMR experiments and was found to be consists primarily of α-Helix and Random coil.The ATP affinity of the protein was measured by fluorescence assay, which uses a fluorescent ATP analog(TNP-ATP), Kd was calculated at 0.303± 0.073 μM.Second, the enzymatic property of the selected peptide was tested through the malachite green assay.The selected peptides exhibits ATPase、UTPase、GTPase、CTPase activities.The most obvious enzymatic property is ATPase. The HPLC experiment was used to analyze the ATP’s hydrolysis product of the protein, and the ATP’s hydrolysis product was found to be ADP. Effect of enzyme’s concentration(20-100 μM) on enzyme activity find that with the increase of the concentration of protein, the enzyme activity increased.The optimum temperature is 45℃, which is higher than the common enzyme, as a result of the original environment have high temperature.The optimum p H is 8.0. The optimum concentration of substrate is 12.5 m M.We find that with the increase of the concentration of Sodium salt, the enzyme activity increased.Finally, we cloned the most prevalent sequence into a plasmid named p ET-28b-abp2. The introduction of Avi-tag makes the protein coding the sequence of single biotinylated lysine sites. Elect its interaction protein in a DNA library which consist of 15 types of reduced amino acids. The most prevalent sequence was itself. Maybe the protein is easy to form oligomers. Throngh the protein crosslinking assay and the protein molecular sieve assay, we found that the protein is exists as a dimer. The results of this study not only helps us to understand the original protein’s physical and chemical properties, but also have a great significance on the origin of protein interaction and the origin of life.Our study is important for basic science and have a potential application value.