核酸酶P1的原核表达、纯化及酶学特性分析

Prokaryotic expression, purification and enzymatic properties of nuclease P1

摘要:

为了建立一种核酸酶P1 (Nuclease P1，NP1) 的原核表达纯化系统，首先采用重叠延伸PCR将22段寡核苷酸拼接，获得人工合成的NP1 基因。将其克隆至分泌型表达载体pMAL-p4X获得重组质粒pMAL-p4X-NP1，然后将重组载体转化T7 Express 和Origami B(DE3) 菌株诱导表达，利用Amylose亲和层析柱纯化获得重组蛋白，并对其活性、热稳定性和金属离子依赖性进行系统分析。SDS-PAGE结果显示，重组蛋白MBP-NP1 (Maltose binding protein-NP1) 在T7 Express 和Origami B(DE3) 菌株中均可表达，且以可溶性形式存在。活性检测表明Origami B(DE3) 菌株中获得的重组蛋白活性高于T7 Express菌株 (75.48 U/mg : 51.50?U/mg)；利用蛋白酶Factor Xa切除MBP标签后，两种重组蛋白的比活力均有提高，分别为258.13 U/mg和139.20 U/mg。重组NP1表现出良好的热稳定性，80 ℃ 温浴30 min后重组酶仍具有90％以上的活力。2.0?mmol/L Zn2+对NP1有比较明显的激活作用，相同浓度的Cu2+则对该酶有强烈的抑制作用。该研究实现了NP1在大肠杆菌系统中的功能性表达，为NP1纯酶的制备提供一个替代途径。

Abstract:

To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 °C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized?the functional expression of NP1 in the prokaryotic system for?the?first?time, and provided an alternative pathway for NP1 preparation.