Methodology of study of MTs

 

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Obtaining in-vivo synthesised Metal-MT aggregates

 

 

Metal-binding to MT is a complex process which can be influenced by Metal-concentration, buffer composition, pH, temperature, redox potentials, etc...

In order to get physiollogically-significant metal-MT aggregates, in our research group, MTs are recombinantly synthesised in E.coli  following a rationale based on the heterologous synthesis of the protein in E.coli cells harbouring the cDNA  encoding for the desired MT, in its wild-type form, in form of splitted domains or site-directed mutants.

E.coli cultures of the recombinant bacteria are supplemented with metals (Zn, Cu or Cd) and the corresponding Metal-MT aggregates are purified by affinity chromatography.

A protocole for cloning of cDNA for heterologous synthesis in E.coli is available at the Tools section

 

 

   

 

 

 

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   Studies of Metal-binding behaviour of the obtained aggregates

 

How do we study Metallothioneins

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Comparative analysis of the spectroscopic, spectrometric and analytic features of the native aggregates and the aggregates resulting from in vitro Cu or Cd titrations of Zn-MT aggregates give us information about its metal-binding preferences, structure and metal-binding abilities.

For each in-vivo synthesis, we obtain completely pure metal-MT aggregates, with a yeild of about 2mg protein/3L culture synthesis, and at concentrations ranging from 0,05mM to 0,2mM. These samples are suitable for analysis by the following techniques: ICP-AES (Inductively-Coupled Plasma Atomic Emission Spectroscopy), CD (Circular Dichroism) and UV Spectroscopy, ESI-Q-TOF (Electro Spray Ionization Quadrupole Time-of-Flight) Mass Spectrometry and CG-FPD ( Gas Chromatography Flame Photometric Detector).

 

 

     

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   Correlation of the observed features with the primary structure of MTs.

 

How do we study Metallothioneins

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The data obtained from experimental analyses of the metal-MT aggregates are considered together with the analysis of the primary structure and MT sequence alignments, in order to define correlations between the sequence motifs and metal-binding behaviour.

 In some cases, generation of mutant forms by genetic engineering confirms the role of specific amino acids and/or domains in metal coordination.

With this final study, we are able to identify the main elements in the constitution of the Metal-MT aggregates and which roles play each element in protein folding, metal preferences, functionality and structure.

 

     

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