Part:BBa_K4023000
Modified MTIA
Overview
Modified MTIA codes for a metallothionein IA protein modified to possess specificity to arsenite in all its 7 metal binding sites. This part is optimized for expression in E.coli. When expressed, the metallothionein IA protein is intended to sequester arsenite entering E.coli. The part can therefore be used in whole cell remediation of arsenite.
Usage and Biology
Metallothionein are small proteins containing a high percentage of conserved cysteine residues. It has 7 metal binding sites, 3 for zinc and 4 for Cadmium and can bind a variety of different heavy metals, with different binding affinity. Metallothionein-IA is a metallothionein protein found in humans. It is broadly expressed in the liver, which matches its known function of detoxification of heavy metals. It is also known to protect against oxidative stress and carcinogens.
Hence, taking advantage of this property, these binding sites were all modified to be more specific to Arsenic. This would potentially increase the concentration of Arsenic accumulated by the protein in a single bacteria cell. Additionally, alterations in geometry and sequence made in the binding sites meant that the protein’s energetic stability was changed. Thus, the protein backbone also needed to be modified to energetically stabilize the protein, particularly when it is bound to Arsenic. As MTIA is a eukaryote gene, the sequence needed to be optimized for expression in E.coli. Hence the gene has been optimized with the IDT codon optimization tool and Benchling, looking out for GC content, uridine content and hairpin loops. Our initial project goal focused on the accumulation of Arsenic from a human gut environment. This resulted in the use of mammalian proteins. The 4MT2 rat metallothionein protein was used as a template model for the MT1A human metallothionein sequence. Despite the project’s transition into an environmental focus, the mammalian protein modeling serves as a proof of concept for modifications made in metallothionein proteins. Our protein modeling work provides a foundation for future work on plant metallothioneins.
The 7 metal binding sites, (3 Zinc and 4 Cadmium) in Human Metallothionein IA have been modified to be more specific to Arsenic. Hence, it can be expected to sequester As III with higher efficacy as compared to wildtype metallothionein-IA.
The MTIA gene has been optimized to express metallothionein IA proteins in E.coli for remediation of As III from the environment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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