Recombinant Expression And Functional Analysis Of Metallothionein MTT1and MTT2from Tetrahymena Thermophila

Metallothioneins (MTs) are low-molecular-weight (6-7kDa), cysteine-rich, metal-binding proteins. MTs are devoted to chelation of toxic metals (Cd, Hg, etc.) and to the regulation of the metabolism of essential trace metals (Cu, Zn, etc.). There is increasing evidence that MTs also act as free radical scavengers. Tetrahymena thermophila has different MT isoforms. Therefore, it provides a good model for metallothionein study. In the study, functions of metallothionein MTT1and MTT2were analysed in vivo, the results are as follows:1. Plasmid pGEX-MTT1and pGEX-MTT2were constructed. pGEX-MTT1and pGEX-MTT2were transformed into Escherichia coli BL21(DE3), respectively. The GST-MTT1and GST-MTT2were expressed after the induction with IPTG. The proliferation of E.coli BL21/pGEX-MTT1is faster than E.coli BL21/pGEX under Cd2+stress. This result showed that MTT1involved in detoxification of Cd2+. However, the proliferation of E.coli BL21/pGEX-MTT2is faster than E.coli BL21/pGEX under normal and Cd2+/Cu2+stress. This result showed that MTT2promote cell metabolism and involved in detoxification of intracellular excess Cd2+/Cu2+.2. To explore the relationship between two subfamilies, the MTT1-MTT3and MTT2-MTT4gene knockout vectors were constructed. MTT1-MTT3and MTT2-MTT4somatic knockout strains were obtained by homologous recombination. Compared with the wild-type strain,△MTT1-MTT3mutant strains are sensitive to Cd2+and△MTT2-MTT4mutant strains are sensitive to Cu2+and H2O2. In△MTT2-MTT4strains, the expression level of MTT5decreased. Under500μM Cu2+inducing, the expression level of MTT1, MTT3and MTT5increased6.0,9.4and8.5times compared to wild type strains, respectively. In△MTT1-MTT3mutant strains, the expression level of MTT2, MTT4and MTT5decreased. Under5μM Cd2+inducing, the expression level of MTT2and MTT4decreased4.9and2.5 times compared to wild type strains respectively, but the expression level of MTT5up-regulated2.9times. The results showed that MTT1, MTT3and MTT5mainly involved in detoxification; in contrast, MTT2and MTT4mainly participated in normal cellular metabolism. The two different MTT subfamily proteins are interactional and have functional compensation in Tetrahymena.