The trypsin inhibitor(LUTI) gene of Linum usitatissimum was inserted into pGEX-6p-1 to construct the recombinant plasmid pGEX-6p-1-LUTI and then was transferred into E.coli strain BL21(DE3),and expressed by IPTG induction.The fusion protein GST-LUTI was efficiently purified by GST-Sepharose4B and digested by GST-Prescission Protease.The digested protein solution were loaded on the GST-GST-Sepharose4B cloumn,then rLUTI was flowed though the GST-Sepharose4B beads,which bound by GST-Prescission Protease and GST,and then polished by Sephacryl S-200.The results indicated that rLU-TI is not only a kind of trypsin inhibitor but also α-glucosidase inhibitor.The inhibition of trypsin and α-glucosidase of rLUTI were higher than natural LUTI' s.The disulfide bonds in rLUTI has siginificantly effect on the inhibitive activity of trypsin and α-glucosidase.Both Cys4 and Cys 49 play a critical role in double enzyme inhibitory activity.C4/A mutaion in rLUTI showed the inhitiory activity of trypsin and α-glucosidase were slightly decreased,but its secondary structure has obviously changed.C49/A or C4、49/A mutaion in rLUTI have lost the inhibitory activity of trypsin and reduced the inhibitory activity of α-glucosidase.These results suggested that the Cys4 of rLUTI has significant effect on the secondary structure and the Cys49 of rLUTI plays an important role in maintaining its double inhibitory activity,especially on the inhitiory activity of trypsin.%亚麻胰蛋白酶抑制剂(Linum usitatissimum L.LUTI)是亚麻中一种兼具胰蛋白酶抑制剂和α-葡萄糖苷酶抑制剂双重活性的多肽.文章将LUTI基因连接在pGEX-6p-1载体上获得重组载体pGEX-6p-1-LUTI,转化至大肠杆菌菌株E.coliBL21(DE3)表达,经GST-Sepharose4B、Prescission Protease酶切GST标签、SephacrylS200凝胶层析,获得分子量大小为8 kDa电泳纯的重组LUTI(rLUTI).经抑制活性测定,相比天然的LUTI,rLUTI对胰蛋白酶和α-葡萄糖苷酶抑制活性明显提升.二硫键存在与否对两种酶的抑制活力均有影响.利用定点突变技术获得rLUTI的C4/A,C49/A,C4、49/A突变体,进一步双酶抑制活性及CD结构分析显示,rLUTI的C4/A、C49/A及C4、49/A突变体对胰蛋白酶抑制活性影响尤为明显,α-葡萄糖苷酶的抑制活性略微下降;其中rLUTI的C49/A及C4、49/A突变体完全丧失了对胰蛋白酶的抑制活性.C4/A的突变对rLUTI的二级结构影响最为明显,导致α螺旋结构含量的降低.以上结果表明rLUTI第4位氨基酸Cys是影响其二级结构的关键位点,第49位氨基酸Cys则在维持其双酶抑制活性上发挥着重要作用,特别在胰蛋白抑制活性方面尤为突出.
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