| 摘要: |
| 【目的】对大肠杆菌噬菌体phiV10第45个基因编码蛋白PNE45进行原核表达，并检测其酶活性。【方法】设计并合成10段密码子优化的寡核苷酸序列，采用重叠延伸PCR技术，将其拼接克隆，获得人工合成的PNE45基因，然后将其整合入含有MBP纯化标签的pET21a原核表达载体中，构建重组载体pET21a-MBP PNE45，诱导表达后获得融合蛋白MBP-PNE45。利用Amylose亲和层析纯化融合蛋白MBP-PNE45，以pUC19为底物，分别在Mg2+和Mn2+参与下对MBP-PNE45活性进行初步鉴定。【结果】融合蛋白MBP-PNE45主要以可溶性形式表达，其表达量占细胞总蛋白的35%左右。PNE45融合蛋白在Mg2+缓冲液中具有DNA随机切刻活性，在Mn2+存在时还具有切刻位点对位切刻活性。【结论】 首次成功表达并制备了PNE45，并初步证明其是一种新的随机性切刻核酸酶。 |
| 关键词: PNE45 重叠延伸PCR 表达纯化 DNA切刻核酸酶 |
| DOI： |
| 分类号: |
| 基金项目:中央高校基本科研业务专项(QN2009070) |
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| Prokaryotic expression and activity identification of DNA nicking endonuclease PNE45 |
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LIANG Yafeng, ZHANG Chao, WANG Yanan
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College of Life Sciences,Northwest A&F University
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| Abstract: |
| 【Objective】In this study,we attempted to express and purify protein PNE45,which is encoded by gene 45 of Escherichia phage phiV10,and further to identify its function.【Method】Using overlapping PCR,10 codon optimized oligonucleotides were spliced to get a synthetic PNE45 gene.The pET21a-MBP-PNE45 expression recombinant vector was constructed.The fusion proteins MBP-PNE45 were purified by Amylose affinity chromatography,and activities identifyication was carried out using pUC19 as substrate in Mg2+ and Mn2+ respectively.【Result】MBP-PNE45 was soluble,and the percentage of fusion protein was up to about 35%.PNE45 exhibited random DNA nicking activity in Mg2+ buffer,while also counter nicking activity in Mn2+ buffer.【Conclusion】The novel random nicking endonuclease PNE45 was expressed,purified and functionally identified. |
| Key words: PNE45 overlapping PCR expression and purification DNA nicking endonuclease |
