Heterologous Expression And Functional Analysis Comparison Of Two Metallothioneins From Tetrahymena Elliotti

Metallothioneins(MTs)are a superfamily of low molecular weight,cysteine-rich proteins which involved in chelating metal ions.Ciliate MTs is diversity and different from that of higher organisms MTs.MTs from Tetrahymena belong to the family 7,which contain repeated different modules.Tetrahymena MT sequence is less conservation,and is longer than that of mammals.The different tandem repeat structure in Tetrahymena MTs could invovled in different resistance to metal ions.In this study,two different isoforms of MT gene Te-MTT2-L and Te-MTT2-S were identified from Tetrahtmena elliotti.The relation between MT sequence length and detoxify ability was analyzed in vivo.The results are as follows:1.Identification of Te-MTT2-L and T-MTT2-S Te-MTT2-L is 447 bp without intron and encodes 149 amino acids;Te-MTT2-S is 273 bp without intron and encodes 90 amino acids.The genes have typical Cu-MTs characterization,belonging to the 7b subfamily.2.Expression Te-MTT2-L and T-MTT2-S in Escherichia coli Recombinant plasmids pGST-Te-MTT2-L,pGST-Te-MTT2-S pSUMO-Te-MTT2-L,and pSUMO-Te-MTT2-S were constructed and transformed into E.coli BL21,respectively.GST-Te-MTT2-L,GST-Te-MTT2-S,SUMO-Te-MTT2-L and SUMO-Te-MTT2-S were expressed from E.coli BL21/pGST-Te-MTT2-L,E.coli BL21/pGST-Te-MTT2-S,E.coli BL21/pSUMO-Te-MTT2-L,and E.coli BL21/pSUMO-Te-MTT2-S at 37? by 0.05mmol/L IPTG induction,respectively.SDS-PAGE analysis showed that SUMO tag can significantly increase the expression of Te-MTT2-L and Te-MTT2-S and improve solubility.Then,SUMO-Te-MTT2-L and SUMO-Te-MTT2-S were purified by affinity chromatography.SUMO-Te-MTT2-L and SUMO-Te-MTT2-S were stable at 85? for 15 minutes.The results showed that SUMO tag is effective for soluble expression of Te-MTT2.3.Comparision of recombinant E.coli resistance to Cu2+ and Cd2+The proliferation of E.coli BL21/SUMO-Te-MTT2-L is faster than other recombinant E.coli BL21/SUMO-Te-MTT2-S under Cu2+ and Cd2+ stress.This suggests that longer MT sequence has a stronger detoxify ability.4.E.coli BL21/SUMO-Te-MTT2-L significantly improved the chelating ability for Cu2+ and Cd2+ Metal ions chelating ability of recombinant E.coli was detected with X-ray fluorescence spectrometer.The level of chelating Cu2+ in E.coli BL21/SUMO-Te-MTT2-L increased 10 times compared to that of BL21/pE-SUMO,increased 1.6 times for Cd2+.Furtherfore,Cu2+ and Cd2+ chelation occurs mainly in 5 hours.The level of chelating Cd2+ in E.coli BL21/SUMO-Te-MTT2-S increased 6.5 times than E.coli BL21/pE-SUMO,increased 1.4 times for Cd2+.The level of chelating Cu2+ and Cd2+ in E.coli BL21/pE-SUMO is lower to that in E.coli BL21/pSUMO-Te-MTT2-L.These results suggest that longer sequence of Te-MTT2-L chelate more metal ions in a shorter time than Te-MTT2-S.In summary,two different isoforms of CuMT gene Te-MTT2-L and Te-MTT2-S were identified from T.elliotti.Te-MTT2-L and Te-MTT2-S were expressed and purified.E.coli/SUMO-Te-MTT2-L not only enhanced copper and cadmium tolerance but also increased metal ion accumulation.The results showed that the longer MTT has a stronger and faster response to metal ions.