灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

Expression, purification and characterization of non-specific Serratia nuclease in Escherichia coli

摘要:

以灵杆菌基因组DNA为模板，PCR扩增非特异性核酸酶 (Non-specific nuclease，NU) 基因，并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21，经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU，MBP-NU)，其最佳诱导表达条件为37 ℃，0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性，在37 ℃、pH 8.0时活性最高，比活力为1.11×106 U/mg，目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid，EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride，PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响，因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。

Abstract:

To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 °C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 °C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11×106 U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.