Construction of α–α domains structure in recombinant monkey metallothionein-1

Monkey metallothionein α domain tandem repeats (4mMTα), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC-10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMTα-EGFP fusion, was constructed and used to target 4mMTα and EGFP on the surface of Pseudomonas putida X4 (CCTCC-209319). The surface location of the INAXN-4mMTα-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMTα on the engineered strains was four times higher than that of the wild-type X4. The Cd²⁺ accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu²⁺ and Zn²⁺. Moreover, the surface-engineered strains could effectively bind Cd²⁺ under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMTα-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMTα-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants.

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Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

Chen

Huang

2011

Appl Microbiol Biotechnol

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The Properties of the Metal–Thiolate Clusters in Recombinant Mouse Metallothionein-4

2005

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Metallothioneins (MTs) are metal-binding proteins with low molecular weight and conservative cysteine residues. Metallothionein-4 (MT-4), one of MT isoforms, is first reported to be distributed in a tissue-specific manner, mainly in stratified squamous epithelia. Here, we compare the properties of metal-thiolate clusters in MT-4 to those in MT-1 and MT-3, including the stabilities toward both pH change and EDTA, as well as the exposure of thiolates to solvent. The metal-thiolate clusters in MT-3 show different property and activity to the reactions compared with MT-4 and MT-1. The structure of metal-thiolate clusters in MT-4 is similar to that of MT-1 from the UV and CD spectra. During pH titration and DTNB reaction, MT-4 and MT-1 exhibit comparable behavior. But while reacting with EDTA, the metal-thiolate clusters in MT-4 are more stable than those of MT-1. We suppose the negative charge of the beta-domain of MT-4 prevents the EDTA attack to MT-4.

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Metalation of a rice type 1 metallothionein isoform (OsMTI-1b)

Malekzadeh

Shahpiri

Siapoush

2020

Protein Expression and Purification

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Construction of α–α domains structure in recombinant monkey metallothionein-1

Cited by 4 publications

References 30 publications

Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

The Properties of the Metal–Thiolate Clusters in Recombinant Mouse Metallothionein-4

Metalation of a rice type 1 metallothionein isoform (OsMTI-1b)

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