Studies On Prokaryotic Expression And Metal Binding Capacity Of Cucumber Metallothionein-like 2 (CsMT-L2)

Metallothioneins (MTs) is a class of low molecular weight, high Cysteine and metal-rich protein. MT was first isolated from horse renal tissue where cadmium accumulate, Metallothionein-like proteins from plants share high homologous with MTs from animals. According to the location and arrangement of Cys, plant MT-like proteins were about categorized into three classes. The biggest differences of class I and class II MT are that Cys of the former concentrated in the peptide N-terminal and C terminal separated by the space region, while, Cys of the latter were scattered throughout the whole squence; and class III MT are not production of genes coding, production of enzyme-catalyzed synthesis as glutathione substrate. Research of structure and function of plant MTs was not fully understood, Mts generally apppear to play key role in metal-rich aspect.CsMT-L2 gene was identified and obtained from cDNA library of cucumber in our laboratory,but the function has been unknow. In this study CsMT-L2 gene was expressed in E coli, metal binding ability of its expression protein was analyzed and compared withβdomain of the human metallothione and phytochelatins, meanwhile, two Cys-rich regions of CsMT-L2 were directly connected to form space region deletion mutant CsMT-L2m, explored function of the space region. The studies will provide a theoretical basis for further revealing relationships between structure and function of MTs, clarify and highlight the role of the space region of I type plant metallothionein-like protein, lay the solide foundation for transgenic plants in future. The main results are as follows:1.Construction of four revelent prokaryotic expressive vectors successfullyFour revelent genes were amplified via PCR using PrimStar HS high-fidelity polymerase, ligated to multiple cloning site region of pET32a+ respectively, after restriction enzyme digestion, sequencing verification, four genes were successfully inserted into pET32a+, the open reading frame are completely correct.2.Four fusion proteins were over-expressed in E coliExpressed fusion proteins were purified by MagneHisTM Protein Purifiction System, analyzed by SDS-PAGE, verified further by S-Tag Western Blot, The expeceted moleculer mass of four fusion protein were oberserved and exhibited.3.Metal binding ability of four fusion proteins were verified in E coli.Binding metal content of four fusion proteins were measured via flame atomic spectrometry, binding Zn2+ capacity of CsMT-L2, CsMT-L2m, HsMTb, PcL fusion protein in vivo bacteria was 1.37×10-5 mol/g,0.693×10-5 mol/g,0.948×10-5 mol/g,0.911×10-5 mol/g, accordingly, Cd2+ adsorption capacities of them is 1.25×10-4 mol/g,3.43×10-5 mol/g,1.15×10-5 mol/g,7.67×10-5 mol/g, the results showed that they were significantly higher than the control (p<0.01), further indicated that the four proteins all confer the ability of binding metal, but their metal-binding capacities exist the significant differences, it may be result from their structures, binding metal ability of CsMT-L2 proteins in bacteria was the most, however, binding capacity of CsMT-L2m decreased significantly (especially Cd2+), it revealed that the space region played an important role in coordinating the folding of two Cys-rich regions.4. The signal peptide, transmembrane region, phosphorylation sites, secondary and tertiary structures of CsMT-L2 and CsMT-L2m were all comprehended.Signal peptide, trans-membrane region, phosphorylation sites, secondary and tertiary structure as well as molecular phylogenetic evolution of CsMT-L2 and CsMT-L2m were predicted and inferred via bioinformatics methods and software. The results showed that CsMT-L2 and CsMT-L2m proteins were non-transmembrane proteins without a signal peptide, CsMT-L2 contains three phosphorylation sites, The secondary structure of CsMT-L2 is consist of the Alpha helix (Hh), Extended strand (Ee) and Random coil (Cc), Alpha helix (Hh), Extended strand (Ee) and Random coil (Cc) accounted for 11.84%,9.21%,78.95% individually. While the secondary structure of CsMT-L2m is all consist of random coil. The three-dimensional model of CsMT-L2 is in accordance with hairpin model.