| 摘要: |
| [目的]克隆小麦种子过氧化物酶基因wp1,并利用原核系统表达纯化.[方法]通过RT-PCR从小麦未成熟种子中扩增wp1基因cDNA,构建His-tag和MBP融合表达载体,并在大肠杆菌中进行表达.使用直链淀粉亲和层析获得MBP-WP1融合蛋白,并以ABTS为底物检测酶活性.[结果]获得的wp1基因cDNA编码一种358个氨基酸残基的蛋白产物,其序列与大麦BP1相似性高达89%;空间结构预测结果表明,小麦WP1属于第三类过氧化物酶,具有该家族成员典型的血红素和钙离子结合位点;His-Tag融合的WP1主要以包涵体形式存在,而MBP融合的WP1主要以可溶形式存在,表达量分别达到总蛋白的18.2%和34.6%;纯化获得的MBP-WP1融合蛋白可检测到过氧化物酶活性.[结论]使用原核系统可高效表达出可溶性的、具有部分活性的WP1蛋白. |
| 关键词: 小麦 种子过氧化物酶 高GC含量 基因克隆 基因表达 |
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| 基金项目:国家自然科学基金 |
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| Cloning and expression of wheat peroxidase 1 (wpl) in E. coil |
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| Abstract: |
| 【Objective】 The study cloned the gene of wheat seeds peroxidase wp1 and obtained this enzyme by expression it in prokaryotic system. 【Method】 The cDNA of Wheat peroxidase 1 ( wp1 ) was amplified from immature seeds by RT-PCR.His-tag and MBP fusion expression vectors were constructed respectively,and fusion proteins were expressed in E.coli strain T7 Expression.The MBP-WP1 fusion protein was purified by amylose affinity chromatography and its peroxidase activity was detected using ABTS as substrate. 【Result】 The cDNA of wp1 codes a protein of 358 amino acids,having a high sequence similarity (89%) with barley peroxidase 1 (BP1).Predicted structure indicates that WP1 belonged to class Ⅲ peroxidase and it had their typical heme binding site and Ca2+ binding sites.His-tag fused WP1 existed as inclusion body and most of MBP WP1 were soluble.The percentage of fusion protein was up to 18.2% and 34.6% respectively.The peroxidase activity of MBP-WP1 was detectable.【Conclusion】 Soluble and partial active WP1 can be expressed in prokaryotic system efficiently. |
| Key words: wheat seed peroxidase high GC content gene cloning gene expression |
