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

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。

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 colt 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/rag）. When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/rag 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 rain at 80 ℃ Zn2＋ （2.0 retool/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.