Preparation Of Cytosine Deaminase APOBEC1 And Its Cofactors In Vitro And Crystal Screen Of Cofactors

This thesis contains two parts.In the first part,the cytosine deamination catalytic enzyme APOBEC1?full name:Apolipoprotein B mRNA editing enzyme,catalytic polypeptide 1,abbreviated as A1?is a member of the APOBEC family.A1 binds to ApoB?Apolipoprotein B?mRNA in the presence of RBM47?RNA binding motif protein 47?and/or A1CF?APOBEC1complemetation factor?and catalytical deamination of the 6666 cytosine(C₆₆₆₆)into uracil(U₆₆₆₆)in a unknown way,then produces a truncated body of ApoB100 named ApoB48,A1 participates in regulating physiological processes related to lipid metabolism,but the micro mechanism is unclear.In addition,A1 also regulates the expression of various cytokine transcripts to participates in tumor lesions,as well as edits the viral genome to inhibit the replication of some retroviruses.For later structural analysis and drug screening needs,the first part is focused on the preparation of APOBEC1,A1CF and RBM47 samples and the initial screening of crystallization conditions We tried a variety of expression plasmids and optimized protein purification methods,found that A1 is usually expressed in the form of inclusion bodies in the conventional expression system.Using the solubilizing tags GST and MBP,adding glycerol or detergent to the buffer,or coexpressing A1 with molecular chaperone pGro7,no soluble A1 protein were obtained.A series of expression and purification methods were further designed and tried to refold inclusion body of A1.However,we still failed.The Pichia pastoris system and the A1mutant were also used,and soluble A1 still was unavailable.Finally,we used plasmid pMAL-c4X-M which contains a weak promoter to express soluble A1 recombinant protein in the E.coli expression system.After simplifying and optimizing the purification steps,pure and soluble A1 was obtained by affinity purification.A1 was found to be dimer,and 3mg of the dimeric A1 was obtained per liter of LB culture of E.coli.We expressed and purified a large quantity of soluble auxiliary proteins through the E.coli expression system.Gel filtration assay showed that they were in a uniform.A1CF is monomer,and RBM47 is dimer.Both of them have nucleic acid binding activity.These work will be helpful for assembling the complex of A1,A1CF,RBM47 with ApoB mRNA and its structural research.In the second part,we tried the functional exchange of non-heme iron?II?and?-ketoglutarate-dependent oxygenases PrhA and dioxygenase FtmOx1.Mutation of PrhA's key sites V150 and A232 possibly leads into its switch into other oxygenases.By replacing PrhA-A232 with the key residue Y224 of FtmOx1,we constructed a PrhA-A232Y pET28a plasmid and tried to transform it into E.coli expression system,dimeric PrhA-A232Y and the tetrameric PrhA-A232Y were obtained.The PrhA-A232Y dimer was selected to interact with substrate preaustinoid A1,some other compounds with similar skeleton.PrhA-A232Y was found not to catalyze the dioxygenation reaction.Thus,it is necessary to continue to screen the substrates and redesign the mutation sites in the future.