Expression, Purification, Crystallization And Preliminary Function Research Of Adenylate Kinase Consisting Of Reduced Amino Acids

Ancient proteins play a major role in the origin of life. Tracing the characters of very ancient proteins represents one of the biggest challenges in the study of origin of life. As we know, some elements of protein, such as architectures, catalytic site of enzymes and so on, is extremely conserved during evolution, so that those can serve as molecular fossils to investigate the charcaters of primitive protein on structures and functions. Some studies showed that primitive proteins depend on exotic small molecules to fulfill their functions, particularly, ATP is required by the most ancient protein and is the earliest ligand bound to proteins. It can therefore be said that the origin of primitive proteins benefited from binding with ATP. Given that, we suggested that the formation of the most ancient proteins was induced or selected by ATP. In order to preliminary explore this hypothesis, we proposed a top-down strategy, which was based on reduced amino acids, desigened a new ATP-binding protein sequence that is totally consisted of early amino acid and preliminary studied on it. The key findings are as follow:(1) The full length cAK gene was designed and synthesized. A high expression E. coli strain pET-28b-cAK was constructed by inserting the cAK gene fragment, which was digested with NdeI/XhoI, into pET28b at NdeI/XhoI sites and transforming the reconstructed vector into E. coli BL21(DE3).(2) The expression conditions of the target protein were optimized, which showed the target protein could overexpress in insupenant. Fusion-protein was also expressed by auto-induction in high-density shaking cultures. By Comparison, the output is eight to ten times as much as that with IPTG-induced. It was purified by two steps IMAC and was digested by Thrombin, at last highly enriched cAK was obtained.(3) We scanned a lot of crystallization conditions for both free cAK and complex cAK. The hanging drop vapordiffusion technique was used, and three crystalline conditions were acquired, thereby a foundation is established for further X-ray diffraction.(4) The studies of circular dichroism (CD) showed that the conformation of cAK changed significantly, when small molecule was added into solution. In addition, due to the protein-small molecule (ADP) interations, the stability of cAK was strengthened, the degradation rate was significantly slower than before.