Klenow(exo-)的表达、活性测定及其在焦磷酸测序中的应用

Expression,Activity Measurement of Exonuclease-Deficient Klenow Fragment and its Application in Pyrosequencing

野生的Klenow聚合酶具有3'-5'外切酶活性,会对引物进行切割而造成焦磷酸测序产生错误的延伸信号。为了制备缺失3'外切酶活性的Klenow聚合酶用于焦磷酸测序反应,该研究全合成了外切酶活性缺失突变的Klenow酶基因编码序列,将其插入到表达载体p ET28a(+)中构建重组质粒,并转化到大肠杆菌中,表达外切酶活性缺失的Klenow酶(Klenow(exo-))。通过优化诱导温度、诱导时间与诱导剂浓度,得到了Klenow(exo-)的最佳表达条件为30℃时诱导表达5 h且IPTG浓度为0.1 mmol/L。利用Ni+柱亲和层析法纯化得到了相对分子质量约为67 000的重组Klenow(exo-)酶,并建立了基于生物发光的酶活测定方法,最终将其用于焦磷酸测序反应。结果表明,制备的重组酶具有良好的聚合酶活性但缺失了3'外切酶活性,可成功测定DNA待测模板序列,为焦磷酸测序技术提供了一个有效的关键酶。

The wild Klenow fragment is the large fragment of E. coli DNA polymerase I , lacking 5＇-3＇ exonuclease activity while having the 5＇-3＇ polymerase activity and the 3 ＇-5＇ exonuclease activity of DNA polymerase I. It was widely used in molecular biology experiment. However, the 3＇-5＇ exonuclease activity of the wild Klenow fragment would cut the sequencing primer when used in pyro- sequencing, causing the false sequencing signals. In order to prepare the Klenow fragment lacking 3＇ exonuclease activity for pyrose- quencing, the coding gene fragment of D424A mutant Klenow with deficient exonuclease activity was synthesized, and inserted into the pET28a（ ＋ ） expression vector to construct the recombinant expression vector, named pET28a（ ＋ ）-Klenow（exo-） ,which was transformed into E. coli ArcticExpressTM to express the exonuclease-deficient Klenow fragment （Klenow（exo-） ）. By optimizing the temperature time of induction, and the inducer concentrations, the optimal expression condition was obtained as cultivating the recombinant strain,inducing with 0.1 mmol/L IPTG for 5 h at 30 ℃. The recombinant protein with molecular mass of 67 kDa was purified by using Ni ＋ affinity chromatography and eluted by buffer containing 300 mmol/L imidazole. The SDS-PAGE analysis demonstrated that the recombinant protein had/a good purity. Then the activity detecting method of Klenow（exo-） based on biolumi- nescent reaction was established. And the activity of Klenow（exo-） was calculated as 1.37 U/μL and the specific activity was 354.12 U/mg. This activity detecting method was free of radiolabel and was expected to be used in determination of other polymerase activity. Finally, the Klenow（exo-） was applied in pyrosequencing. The results showed that the Klenow（exo-） had a good polymerase activity and no 3＇ exonuclease activity, and could be applied in the pyrosequencing to sequence a single-strand DNA template. Our study provides an effective key enzyme for pyrosequencing.