Metallothionein Expression In Prokaryotes And In Environmental Governance

Metallothioneins (MTs) are a family of low-molecular-weight, cysteine-rich, metal-binding proteins, widely distributed in a variety of organisms ranging from bacteria to mammals, and are generally considered responsible for heavy metal detoxification and essential metal metabolism in eukaryotes. A predominant feature of metallothionein is that approximately one-third of the amino acids are cysteines, yet the protein contains no disulfide bonds. Metallothioneins' special structure and important function provoke the interest of many scientists. In all kinds of MTs, human MT is reported to be most capable of detoxification. Human MT will be safe in food and cosmetic.In this study, human hepatic MT-I A was chosen. Human MT was expressed in Escherichia coli, the characteristics and the application of the protein was studied in E.coli. It is reported that the rates of production of MTs in E.coli. were low compared with those of other eukaryotic gene products, and the reason is unknown. The metallothionein was extracted from mammalian hepatic traditionally which is expensive and low yielded. Since metallothioneins are useful in medicine, food and cosmetic, they can not be used widely. I tried to produce MTs by fermentation which is a cheaper way.The first part of my work is to design the synthetic gene for hMT-I A. The DNA sequence for the synthesis was based on the amino acid sequence of human hepatic MT_ I A, which is reported in Genbank. The fusion proteins' expression would be affected by codon usage, especially in the heterogeneous express systems. Some codons were changed according to E.coli preferred codons.The designed gene was synthesized chemically. In order to insert the hMT-I A into the plasmid vector, suitable restriction sites of BamH I in N-terminal codon and EcoR I and Sal I in C-terminal codon were included in the synthesized gene sequence. A his-tag was also designed in c-terminal codon.The second part of the study is to construct the expression vector for MT's. Considering the susceptibility of metallothionein to proteolytic digestion, it has been suggested to stabilize MT with fusion tags such as glutathione S-transferase and β-galactosodase, and make sure the structure and function are not affected by the fused proteins. A GST gene fusion system that is called pGEX-4T-l was used. The system is a versatile system for the expression, purification, and detection of fusion proteins produced in E.coli. The synthesized gene products were digested with BamH I and Sal I and then were inserted into vector pGEX-4T-1, which was cleaved with the same restriction enzyme. The constructed plasmid was named pGHM. I have tried several E.coli. strains to express the fusion protein. E.coli. BL21 (DE3) expressed the most targeted protein but the yield is not stable. And the express yield would decrease generation by generation. Concerning the plasmid extraction, E.coli. DH5α is more convenient. So E.coli. BL21 (DE3) was used as the express strains and E.coli. DH5a was used to conserve the plasmid. The experiment results were detected by agarose gels, SDS-PAGE and western blotting.In the third part, As-binding properties of MT fusions were studied. Arsenic is an extremely toxic metalloid that adversely affects human health. There is a variety of methods currently available for removal of arsenic from contaminated water. Biological methods are the cost-effective technology for heavy metal remediation. The engineered bacteria is supposed to accumulate As from water waste. The potential of genetically engineered E.coli BL21 to accumulate arsenic in aqueous solution containing arsenic and organic matter was investigated. To ensure the accuracy of the result, the samples were washed by HEPES buffer and then lyophilized. As concentration was measured by atomic fluorescence spectrometry.The arsenic absorbing capability increased more than three-fold from 76.3 to 319.6 μg/g dry cells of the recombinant E. coli compared with the control. Arsenic bioaccumulation was optimal in low pH while was inhibited by the heavy metals of Cd, Hg, Zn. The optimal arsenic concentration for bioaccumulation was 50 μM, and 70% of the maximum arsenic accumulation per gram of cells was reached within 1 h of the bioaccumulation process. This recombinant strain expressing hMT-I gene will be useful in bioremediation of arsenic contaminated waste water.