非限制性内切核酸酶Sma的表达纯化工艺及性能研究

doi:10.13523/j.cb.20171112

中国生物工程杂志

2017, Vol. 37

Issue (11): 89-93 DOI: 10.13523/j.cb.20171112

技术与方法

非限制性内切核酸酶Sma的表达纯化工艺及性能研究

徐一帆, 刘明秋

复旦大学生命科学学院上海 200438

Expression and Purification Procedure of Nonspecific Endonuclease Sma and Its Performance Study

XU Yi-fan, LIU Ming-qiu

School of Life Sciences, Fudan University, Shanghai 200438, China

全文: PDF(533 KB) HTML

摘要： 目的：大量表达非限制性内切核酸酶Sma并获得高纯度目的蛋白，对其酶活进行鉴定。方法：PCR获得Sma基因片段，构建pET28a-ompA-Sma表达载体，转入E.coli BL21（DE3）中，筛选出不同培养基下，目的蛋白可溶表达量最高的条件。通过渗透休克方法提取目的蛋白，并经离子交换纯化。检测不同的温度条件下Sma的酶活，并与商品化产品进行比较。结果：经PCR和测序证明重组蛋白表达质粒构建正确。可溶蛋白产量为7mg/L，每升培养基获得7 300kU的Sma，纯化后纯度>95%，活性达273U/μl（商品化产品为250U/μl）。结论：成功地表达了可溶性非限制性内切核酸酶Sma，纯度高、活性好，各项条件下活性皆不低于商品化产品。

关键词： 分泌表达; 酶活; Serratia marcescens; 非特异性内切核酸酶; 无机盐培养基

Abstract: Objective:To express nonspecific endonuclease Serratia marcescens (Sma), gain the high purity expressed product, and determine its activity. Methods:Sma fragment was produced by PCR. The constructed recombinant plasmid pET28a-ompA-Sma was transformed into E. coli BL21(DE3) to express soluble and high-yielding Sma by optimizing different medium. Target protein was extracted by osmotic shock, purified by ion exchange. Compared Sma with commercial product by activity test of different temperature. Results:PCR and sequencing proved that recombinant plasmid was constructed correctly. The recombinants Sma at an expression level of 7 mg/L, gained 7 300kU Sma per liter media, super-reached a purity of 95% and a specific activity of 273U/μl (commercial product is 250U/μl) after purification. Conclusion:Sma was successfully expressed. The purified Sma showed a high purity and activity. Under various conditions, the activity is not lower than that of commercial products.

Key words: Minimal medium Activity Serratia marcescens Nonspecific endonuclease Secretory expression

收稿日期: 2017-06-06 出版日期: 2017-11-15

ZTFLH:

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通讯作者: 刘明秋 E-mail: liumq@fudan.edu.cn

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引用本文:

徐一帆, 刘明秋. 非限制性内切核酸酶Sma的表达纯化工艺及性能研究[J]. 中国生物工程杂志, 2017, 37(11): 89-93.

XU Yi-fan, LIU Ming-qiu. Expression and Purification Procedure of Nonspecific Endonuclease Sma and Its Performance Study. China Biotechnology, 2017, 37(11): 89-93.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171112 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I11/89

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