Study Of Refolding And Properties Of Bacterial Expressed RADTZ

Purpose:The mycotoxin aflatoxin B1(AFB1) is an improtant carcinogenic,mutagenic and genotoxic substance.Our institute has screened out a fungus named E20 that is capable of producing an aflatoxin detoxifizyme(ADTZ) and succeeded in cloning and expression of recombinant ADTZ(rADTZ).rADTZ is a new oxidase that has low sequence homology with any known protein.Therefore,the study is significantly theoretical with variable cations. Previous work has cloned this gene in the pMAL-C2X vector,with rADTZ fused to MBP,and succeeded in induced expression in E.coli,with incredible expression levels but low enzyme activity.In this thesis,we investigated the refolding of bacterial expressed and urea-denatured rADTZ with diverse conditions,in order to obtain highly pure and active rADTZ proteins for further structural studies of this protein.Methods:Purifition MBP rADTZ with affinity chromatography followed by Factor Xa cleavage to obtain a pure mixture of MBP and rADTZ.The denatured proteins was refolded under various conditions,including:acidity/basicity,oxidation/reduction,saccharides,amino acids,inorganic salts,surfactants,etc.Evaluated the resultant proteins with HRP detection system.After purified active proteins with Superose^TM 12 10/300 GL gel filtration,the appearance molecular weight of the highly active renatured rADTZ was consequently determined.The secondary structure was determined by using circular dichroism(CD) and the possible metal ion constituent was did by using isothermal calorimetry(ITC) in order to decipher its mechanism.Results and discussion:After various refolding conditions screens,optimized conditions are as followed:7.3μg denatured rADTZ per group in 0.02M Na₂HPO₄-0.01M citric acid(pH5.8) at 4℃under two conditions:â‘ [GSH]/[GSSG]=1:1,with additives of 0.1M NaCl+10%glycerol+Arg/Glu(50mM/10mM)+0.05M Pro,when no EDTA is present;andâ‘¡[GSH]/[GSSG]=3:1,with additives of Arg/Glu(50mM/10mM)+0.05mM Zn when EDTA is present.The renaturing efficiency of the two methods were 63%and 39.6%respectively,enzyme activity were 135U/mL and 85U/mL respectively.The possible mechanism of refolding was:salt increased protein solubility and reduced protein aggregation during renaturing;amino acids helped to prevent aggregation of heat-denatured protein and induced the folding of the elongated peptide intoα—helices and the correct tertiary structure.After purified active proteins with Superose^TM 12 10/300 GL gel filtration,the highly pure rADTZ was obtained,its appearance molecular weight is 76KD,demonstration the active rADTZ is a mononar;the resultant of rADTZ circular dichroism(CD) in extreme ultraviolet field is:α—helix 39.4%,β-sheet 10.2%,β—turn 13.7%and Random coil 36.7%,in accordance with the bioinformation prediction;by using isothermal calorimetry(ITC) we deduced that the zinc ion(s) had an binding site with rADTZ,this has confirmed the bioinformation prediction of a possible exist of zinc finger in ADTZ which with a potential function of hydrolysis.From the experimental results and the former work,suggest that ADTZ possiblely be a bis-functional enzyme with oxidation and hydrolysis;the two cysteine locate at 20 and 502 don't form disulfide bridge both inter- or intra-molecule;the 502nd cysteine lies nearby the zinc finger domain and have a Zn²⁺ binding site.