Study On The Structure And Function Of AIFM2 And Preparation Of C-di-AMP At Large-scale By Enzymatic Reaction

PART IBACKGROUNDApoptosis is an active,orderly,multi-gene regulated cell gradual death process,which is one of the basic phenomena of life.Apoptosis can be divided into two major categories,including caspase-dependent and caspase-independent apoptosis.At present,the study suggest that the apoptosis-inducing factor AIF plays a key role in the caspase-independent apoptotic pathway.1.AIF is a flavin-binding protein localized to the mitochondrial intermembrane,which is transferred from mitochondria to the nucleus under the stimulation of specific apoptosis-inducing signals to cause chromatin condensation and DNA fragmentation2.At the same time,a protein called AMID,which can also induce caspase-independent apoptosis in cells,have been identified as AIF-homologous flavoprotein.(1)Although AIFM2 lacks the mitochondrial localization sequence fragment in comparison with AIF,it still has a NADH oxidoreductase region highly homologous to bacterial oxidoreductase,which can bind FAD and NADH.(2)AIFM2 is mostly located in the mitochondrial outer membrane and a few are located in the cytosol.The effect of AIFM2 on nuclear and chromatin is similar to that of AIF,but its induced apoptosis cannot be inhibited by bcl-2.The specific mechanism of apoptosis is not clear.In order to explore the structural basis and molecular mechanism of AIFM2’s biological function,we conducted a preliminary study on the structure and function of AIFM2.RESULTS1.We have established the methods to overexpress AIFM2,full-length and mutants(including truncations),in prokaryotic expression system,as well as purification methods and we have obtained a high purity and stable target protein.2.In addition,for the smooth acquisition of protein crystals,we selected and purified the AIFM2 homologous protein by the same method,and then obtained a better single crystal of homologous protein by crystal growth and optimization.PART ⅡBACKGROUNDCyclic di-AMP is a bacterial second messenger nucleotide.1.It is involved in many cellular activities such as potassium homeostasis,DNA repair,biofilm formation,central metabolism,virulence and regulation of gene expression.2.It is also valuated as a potential vaccine adjuvant candidate.Accordingly,it is still strongly intensified to develop a more simple and economical preparation method for c-di-AMP.RESULTS1.In this paper,we establish a practical enzymatic method for the preparation of c-di-AMP using Vibrio cholerae dinucleotide cyclase DncV.First,we confirmed that DncV can efficiently catalyze the synthesis of c-di-AMP without obvious product inhibition in vitro.Our enzymatic preparation method mainly includes four steps:(1)The His-tagged DncV recombinant protein is specifically bound to the Ni-NTA resin;(2)The c-di-AMP is efficiently synthesized by using the DncV protein immobilized on the resin;(3)One-step purification by using macroporous absorption resin SP207;(4)The collected solution was subjected to ammoniation neutralization and rotary drying.2.By this method,under the optimum conditions,in which 300 mL of 300 mM NH4Ac/NH3 pH 9.5 buffer containing 20 mg DncV immobilized on 12.5 mL Ni-NTA resin,20 mM MnCl2 and 10 mM ATP was incubated overnight at 30 ℃,and then the synthesized c-di-ATP was one-step purified by using 400 g macroporous absorption resin SP207,we obtained nearly 1 g of the diammonium salt of c-di-AMP of weight purity ≥98%was obtained as a white powder,which corresponds to an overall yield of-82%,based on the ATP input into the reaction.Our experimental results show that this method is superior to all the reported methods and can be easily used in industry to prepare the c-di-AMP on a large scale.