Expression Of TrxA-EstB1 Fusion Protein And Its Purification, Characterization Of Enzyme Activity

The use of insecticides does not only bring benefit to human beings but also cause serious environmental pollution. Many insects including Culex pipiens can product detoxificase that can catalyze and degrade many xenobiotic agents besides pesticides, so it becomes harder to kill them. But the use of insect detoxificase provides a novel method in bioremediation of pesticides contamination ,so this thesis is aimed at fusion expressing the detoxificase(esterase B1) at high level. It will pave the way for utilization of esterase B1 in reality. The DNA fragment of esterase B1 is cloned to MCS of plasmid pThioHisA to construct the thioredoxin(TrxA) and esterase B1 fusion expression vector . The new recombinant plasmid is named pThioHisA-B1. The process is as follows : first, the plasmid pRL-B1 and pThioHisA are digested with restriction endonuclease EcoRI. Then retrieve and purify the 1.33Kb fragment of pRL-B1 and 4.4Kb fragment of pThioHisA through low-melting-point agarose gel electrophoresis. After the purified expression vector fragment is dephosphorylated , link up the two fragments with T4DNA ligase in vitro . After transferring the new recombinant plasmid pThioHisA into the host E.coli DH5αthe positive clones are screened on LB+AMP medium plate with BglⅡand BamHI.The engineering strain E.coli DH5α/pThioHisA-B1 is cultured in LB +Amp medium to an OD550 of 0.5 at 37℃,180rpm ,then add IPTG to a final concentration of 1mmol/L, 12 hours for induction . Higher levels of expression of TrxA-EstB1 is obtained 7 hours after induction . The expressed fusion protein with molecular weight of about 63KD is about 48.7% of the total bacterial protein as detected by SDS-PAGE and thin-layer gel scanning analysis.The bacterial pellet is sonicated and centrifuged at 10000rpm for 20 minutes at 4℃ to pellet cell debris and insoluble matter .The SDS-PAGE confirms that the TrxA-EstB1 fusion protein exists in both inclusion body and soluble protein in the cells. The amount of the soluble target protein increases along with decrease of induction temperature and amounts to more than 30% of the total bacterial proteins at 28℃.Under shake incubator the IPTGconcentration is studied ,The result shows:0.4mmol/L IPTG can induce the E.coli express fusion protein and has no obvious difference when which is 1.0mmol/L.The results of detoxifing experiment indicate that the TrxA-ExtB1 fusion in engineering bacterial of E.coli DH5α/pThioHisA-B1 exhibit a high detoxificase activity in degrading the specific substrate such as α-naphthyl acetate(α-NA).This result shows that the achieved fusion protein keeps the enzyme activity of esterase B1 because the fusion chaperon of TrxA can promote the correct fold of esterase B1 and its soluble expression . We have immobilized the bacterials in 2.5% algmate and 3% CaCl2 in order to make more practical use of the engineering bacterial .It is found that the immobilized cell can work well in degradingα-NA also .The fermented broth is centrifugated at 10000rpm to collect cell pellet, then the pellet is sonicated in lysate buffer and centrifugated to separate the crude fusion protein. The crude fusion protein is purified with ammonium sulphate precipitation,DEAE-sepharose CL-6B anion-exchange and SephadexG-150 gel filtration chromatographies. The purified TrxA-EstB1 protein is demonstrated to be one single protein band by SDS-PAGE. With respect to optimal temperature and optimal pH,substrate specificity,the purified fusion esteraseB1 exhibited properties similar to those of the wild -type esteraseB1. The optimal reaction temperature and pH are 40℃ and 7.5 ,respectively.