High-level Expression, Purification Of TAT-MT Fusion Protein In Escherichia Coli And Bioactivity Assay In Vitro

Objective:Expressing human metallothionein-1A(MT-1A) and Tat-Protein Transduction Domain (Tat-PTD) fusion gene Tat-MT in Escherichia coli,purifying and authenticating target protein TAT-MT, and then detecting its chelating ability to metalions.Methods:The human metallothionein (MT) gene fused with trans-activating transcriptional activator (Tat) was amplified by PCR and inserted into the expression vector pET-3c.The recombinant plasmid was transformed into E.coli BL21 (DE3) plySs, the recombinants were selected by SDS-PAGE. To find out the optimize expression conditions of the targe proteins TAT-MT, tempreture, carbon source, nitrogen source were used to screen expression condition. The Ni-NTA Resin was used to purify the targe protein.The chelating ability to metal ions of Engineering Strain was analysed by atomic absorption spectrometry.Results:The fusion protein TAT-MT was expressed at 20℃for 20h, with IPTG at the final concentration of 1mM induction.Thes size of the target protein was about 15Kd by SDS-PAGE analysis.The wet weight of cell was up to 30g/L in the experiment of small scale fermentation(5L),recombinant TAT-MT took up to 18% of the total bacterial proteins.After harvesting cell by centrifugation and ultrasonication,the target protein TAT-MT was purified using Ni-NTA Resin affinity chromatography, the purity was over 85%. Atomic absorption spectrometry showed that the ability of cadmium and copper ion chelating in recombinant strain improved by 2.5 and 1.27 compared to control empty strain, the difference was significantly(P<0.05).Conclusion:The fusion protein was soluble expressed in Escherichia coli, the fusion protein had resistant potency to the heavy metal by binding to the metalion.