Dissertation > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology

Expression and Purification of Metallothionein from Sinopotamon in the Sumo Fusion System,and Studies on Its Metal Chelating Ability

Author WuYang
Tutor WangLan
School Shanxi University
Course Hydrobiology
Keywords freshwater crab Sinopotamon henanense Metal lothioneinprokaryotic expression vector the SUMO expression system recombinant purification metal chelating capacity
CLC S917.4
Type Master's thesis
Year 2012
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Metallothionein (MT) is a kind of unique protein widely distributed in organisms that is rich in cysteine and with a low molecular weight(2~7kDa). It has no aromatic amino acids or histidine and can be combined with metal. The study in recent years shows that, MT can take part in the storage, transport and metabolism of trace elements, resist to ionizing radiation, clear the free radicals and have a role in the detoxification of heavy metals. The MT of crustaceans is found relatively late.Studies have shown that MT can absorb heavy metals and clear reactive oxygen species by a large number of cys residues, which protects animals from oxidative damage. MT is an important functional genes, which takes part in the development of animals, resistance to stress and other physiological processes. Our group has systematically studied relation of major organizations of crab Sinopotamon, condition of MT expression and grand total of heavy metals. We have also studied of the cloning of full-length sequence MT cDNA induced by cadmium in Sinopotamon henanense. On this basis, this study used PCR amplification, added restriction sites to cloned MT genes and at both ends. The MT gene is cloned into the vector pET28a vector pET28a-SUMO and pGEX-6p-1three expression vectors By Recombinant DNA technology and Constitute a reorganization of expression vector. The sequencing of the company’s sequencing results show that the three recombinant expression vectors were constructed successfully.Our group made three recombinant expression vectors transformed into E. coli BL21(DE3) expression strain, induced expression of the target protein, and collected the supernatant of bacterial ultrasonic broken. SDS-PAGE analysis shows that,IPTG successfully induced MT protein expression. According to SDS-PAGE electrophoresis results, comparative analysis of MT expression in the three recombinant plasmids, and ultimately determined the soluble expression of MT in the SUMO fusion expression system higher. To optimize the the the SUMO prokaryotic expression system induced expression conditions, by studying the induction temperature, induction time and induction of the three conditions on the concentration of MT fusion protein expression levels. Finalized at37℃,1mmol was induced by IPTG for6hours, MT fusion protein soluble expression of the highest. SUMO expression system acquired fusion protein of MT. Because it carries the his label, Ni affinity chromatography was used to purify MT fusion protein. In the process of purification, after the elution of imidazole buffer to collect the purified protein. By SDS-PAGE electrophoresis analysis showed that better purification can remove most of the hybrid protein, pure MT fusion protein.In this study, using site-directed mutagenesis techniques, the primary structure of MT by the line part of the transformation, constructed two mutants of the SUMO-MTt1SUMO-MTt2, and induced the expression of SUMO-MTt1, SUMO-MTt2, SUMO-MT and GST-MT four fusion protein. A final concentration of300μM of Cu, of Cd and Zn in three kinds of heavy metal ions were added in the induction process. After induction, in expression of bacterial, adsorption capacity of recombinant MT to Cu, Cd and Zn in three heavy metals by flame atomic absorption spectrometry. The results show that, compared with the empty bacterial, four kinds of MT fusion proteins of Zn2+and Cd2+and Cu2+three kinds of heavy metal ions have very obvious ability of binding. But each MT fusion protein ability of binding of different metal ions is different. The overall point of view, four MT fusion protein maximum uptake, Cd2+, Zn2+and Cu2+absorption have the similar ability. From four proteins on the same kind of metal ion uptake can be seen, a structural transformation of the mutant proteins are stronger than those mutants not mutation combined with. Especially in the ability of binding to Cd2+, SUMO-MTt1which has been transform significantly improved. This may be related to the number and location of Cys, the length of the intermediate connection zone, It also shows that in both ends of the Cys-rich region, the intermediate connection zone in the conformation of binding metal ions may play an essential role.

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