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-omp A-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 300k U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。
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 highyielding 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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第11期89-93,共5页
China Biotechnology
