关键词: 猪, 金属硫蛋白-1A, 金属硫蛋白-2A, 生物信息学分析, 原核表达, 金属结合特性

Abstract:

【Objective】 The study mainly made the bioinformatics analysis, carried out prokaryotic expression, and explored the metal binding characterization of Sus scrofa metallothionein-1A (SsMT-1A) and metallothionein-2A (SsMT-2A) with Zn(Ⅱ) and Cu(Ⅰ), so as to provide a theoretical basis for the study of the mechanism of action of adding Zn and Cu in diet inducing porcine metallothionein expression, thereby regulating the Zn and Cu homeostasis in vivo, and then promoting the porcine production performance. 【Method】 Firstly, the porcine gene sequences of SsMT-1A and SsMT-2A were obtained from NCBI. Afterwards, their protein sequence characteristics were analyzed by ClustalX2 software and ExPASy database, and then the phylogenetic tree of MT protein molecular was constructed between them and other species using the Mega-X. Secondly, the prokaryotic expression vector pET-28a-SUMO-SsMT-1A/SsMT-2A were constructed and verified by PCR, double digestion, and gene sequencing. Subsequently, the recombinant plasmid was transformed into BL21(DE3) plysS and the expression of SsMT-1A and SsMT-2A was induced using the IPTG. Then, the SUMO-SsMT-1A/SsMT-2A recombinant protein were purified by Ni-NTA affinity chromatography and Superdex-75 column, and were analyzed by Western-blot. In the end, the properties of SsMT-1A and SsMT-2A binding Zn(Ⅱ) and Cu(Ⅰ) were surveyed by the tolerance analysis of Escherichia coli containing SsMT-1A and SsMT-2A genes, circular dichroism (CD), matrix assisted laser analytic ionization time of flight mass spectrometry (MALDI-TOF-MS), and isothermal micrometer calorimetry (ITC). 【Result】 Bioinformatics analysis showed that the protein sequences of SsMT-1A and SsMT-2A were highly homologous, and their cysteine (Cys) content and arrangement motifs were perfectly consistent, and there existed only 8 amino acid residues discrepancy. Through the prokaryotic expression, purification, and SUMO enzyme digestion, the SsMT-1A/SsMT-2A fusion protein were successfully obtained. The tolerance analysis of metal demonstrated that compared with SsMT-2A, E. coli containing SsMT-1A genes had the stronger resistance to Zn and Cu. The CD spectrum illustrated that SsMT-1A and SsMT-2A could combine with Zn(Ⅱ) and Cu(Ⅰ) and both of them exhibited the preference with Zn(Ⅱ) coordination. But SsMT-1A exhibited the stronger binding ability with Zn(Ⅱ) than SsMT-2A. MALDI-TOF-MS results showed that the experimental molecular weights of apo-SsMT-1A and apo-SsMT-2A were respective 6 047.5 Da and 6 048 Da, and the stability order of both of them was and Cu(Ⅰ)> Zn(Ⅱ). ITC results showed that the affinity constants of SsMT-1A and SsMT-2A coordinating Cu(Ⅰ) were higher than that of Zn(Ⅱ), and the stoichiometries of both of them binding Cu(Ⅰ) were 7, while the stoichiometry of SsMT-1A binding Zn(Ⅱ) was 2. 【Conclusion】 Although SsMT-1A and SsMT-2A possessed the high homology, the difference of 8 amino acid residues determined their different binding feature with Zn(Ⅱ) and Cu(Ⅰ). Although SsMT-1A and SsMT-2A shared a highly consistent binding behavior of Zn(Ⅱ) and Cu(Ⅰ), their characteristics of binding Zn(Ⅱ) and Cu(Ⅰ) was slightly different. Thus, they should not be considered as completely physiological equivalent molecules. According to the relationship of structure and function of MT, SsMT-1A might play a role in detoxification of heavy metal ions and SsMT-2A might mainly regulate Zn homeostasis. The study laid a foundation for further elucidating the role of SsMT-1A and SsMT-2A in regulating metal ion homeostasis to promote pig production performance.

Key words: Sus scrofa, metallothionein-1A, metallothionein-2A, bioinformatics analysis, prokaryotic expression, metal binding characteristic