黄曲霉毒素解毒酶在大肠杆菌中的可溶性表达、纯化及其圆二色谱分析

Soluble Expression,Purification of Recombinant Aflatoxin-detoxifizyme in E.coli and Analysis of Circular Dichroism Spectrum

目的:应用原核表达系统对黄曲霉毒素解毒酶(aflatoxin-detoxifizyme,ADTZ)进行高效可溶表达和纯化,并对其进行生物学活性与二级结构分析。方法与结果:亚克隆ADTZ的成熟肽,并构建与pMAL-c2x的重组质粒,转化大肠杆菌Rosetta(DE3),IPTG诱导实现了MBP_ADTZ融合蛋白的高效可溶表达,其表达量约占总蛋白的50%。经Amylose亲和层析、Factor Xa酶切和疏水层析后得到高纯度的rADTZ蛋白。生物学活性分析表明rADTZ蛋白具有降解AFB1的酶活性,酶比活为136U/mg。圆二色光谱对rADTZ蛋白二级结构的分析结果为:α-螺旋为43.3%、β-折叠为31.1%、β-转角为10.5%和无规则卷曲为15.1%。结论:用大肠杆菌成功表达并得到高纯度有活性的rADTZ蛋白,为进一步对rADTZ结构与功能研究奠定了基础。

Objective： To expresss and purify the aflatoxin-detoxifizyme（ADTZ） in E.coli.and to study the biological activities and secondary structure of rADTZ.Methods and Results： the mature peptide of ADTZ was subcloned into pMAL-c2x vector to construct the prokaryotic expression plasmid.The recombinant plasmid was transformed into Rosetta（DE3） and the soluble fusion protein MBP-ADTZ was highly induced by IPTG.The expression level was approximately 50% of the total bacterial protein.The pure fusion protein was got from the cell lysate by amylose affinity chromatography.42kDa MBP and 76kDa rADTZ were gained via digestion of fusion protein by Factor Xa,and pure rADTZ was obtained by Hydrophobic interaction chromatography（HIC）.Biological activities of rADTZ had been examined and results showed that the protein had AFB1-detoxifying activity with the specific activity of 136U/mg.Circular dichroism spectra analysis revealed that rADTZ was composed of 43.3% of α-helix,31.1% of β-sheet,10.5% of β-turn and 15.1% of random coil.Conclusion： Purified rADTZ protein with AFB1-detoxifying activity was obtained and that laid the foundation for exploration of the relationship between the structure and function of rADTZ.