Kod DNA聚合酶的制备及纯化研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1210

摘要/Abstract

摘要：

Kod DNA聚合酶作为常见的高保真DNA聚合酶,具有较强的DNA延伸能力以及3'-5'外切核酸酶校正活性。本研究通过对重组Kod DNA聚合酶分离纯化条件的优化,以期提高其表达和纯化效率以及片段扩增能力。利用异丙基硫代-β-D-半乳糖苷（isopropyl β-D-thiogalactoside,IPTG）对pET-30a-Kod进行诱导表达后,利用酶溶法和超声波破碎法对细胞破碎并提取粗酶液,然后利用Ni-NTA柱洗脱纯化获得较纯Kod DNA酶,再经透析得到高纯度Kod DNA酶,利用PCR反应和Sanger测序手段来检测自提纯化的Kod DNA聚合酶活性及保真性。同时,本研究对IPTG浓度和洗脱液中咪唑浓度等重要参数进行优化。结果表明,在28℃条件下0.1 mmol/L IPTG诱导16-18 h后,Kod DNA聚合酶高效表达;当咪唑浓度为200 mmol/L时蛋白洗脱效果最佳。PCR反应体系中Mg²⁺浓度为1.5 mmol/L,Kod DNA聚合酶浓度为0.061 25 μg/µL时,Kod DNA聚合酶效率较高,能有效地扩增3 194 bp的目的条带;通过序列比对,PCR产物序列中未引入任何突变。经优化制备后的Kod DNA聚合酶在酶活性和扩增保真性上均能达到商用高保真DNA聚合酶水平。本研究为节省实验室PCR成本,进一步开发和利用Kod DNA聚合酶奠定一定的基础。

关键词: DNA聚合酶, 聚合酶链式反应（PCR）, Kod DNA聚合酶, 蛋白质纯化

Abstract:

Kod DNA polymerase,as a common high-fidelity DNA polymerase,has strong DNA extension ability and 3'-5' exonuclease activities. This study is aimed to improve the expression efficiency and extension ability as well as fragment amplification ability by optimizing the isolation and purification conditions of recombinant Kod DNA polymerase. First,isopropyl β-D-thiogalactoside（IPTG）was used to induce pET-30a-Kod for expression,and enzymatic dissolution and ultrasonic fragmentation were to grind the cells and extract the crude enzyme solution,then Ni-NTA column was to elute and purify the Kod DNA polymerase. Finally,the high-purity Kod DNA polymerase was obtained by dialysis. PCR and Sanger sequencing were used to detect the activity and fidelity of self-purified Kod DNA polymerase. Meanwhile the key parameters such as IPTG and imidazole concentrations were optimized. The results showed that Kod DNA polymerase was highly expressed after induced by 0.1 mmol/L IPTG at 28℃ for 16-18 h,and the eluted effect was optimal while imidazole concentration was 200 mmol/L. The efficiency of the Kod DNA polymerase was the highest while Mg²⁺ was 1.5 mmol/L and Kod DNA polymerase was 0.061 25 μg/µL in the PCR reaction system,under which 3 194 bp target band was efficiently amplified;and there was no introduced mutation in the PCR product after sequence alignment. The enzymatic activity and fidelity of self-extracted Kod DNA polymerase after optimized preparation reached the same level of commercial high-fidelity DNA polymerase. Thus,this study lays a foundation for saving the cost of laboratory PCR and further developing and utilizing Kod DNA polymerase.

Key words: DNA polymerase, polymerase chain reaction（PCR）, Kod DNA polymerase, protein purification

易芳, 来鹏程, 郑希鳌, 胡帅, 高燕丽. Kod DNA聚合酶的制备及纯化研究[J]. 生物技术通报, 2022, 38(5): 183-190.

YI Fang, LAI Peng-cheng, ZHENG Xi-ao, HU Shuai, GAO Yan-li. Research on the Preparation and Purification of Kod DNA Polymerase[J]. Biotechnology Bulletin, 2022, 38(5): 183-190.

图/表 6

表1 PCR扩增引物信息

Table 1 Information of the primers used for PCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
AtVSR6 gDNA-F	ATGAGTCTCCAACCAATGAGAC	3 194
AtVSR6 gDNA-R	ACATTTCCCCAACCAATTCATTC- TCTGT	3 194

图1 不同浓度诱导剂IPTG对Kod DNA聚合酶表达的影响不同浓度IPTG对Kod DNA聚合酶表达的影响。M：蛋白分子质量标准;A：0.1 mmol/L IPTG诱导Kod DNA聚合酶表达。1：Kod DNA聚合酶粗提液（CSKod）;2：超声破碎后离心的残渣重悬液（CMKod）;3：透析后经储存缓冲液1∶1稀释后的Kod DNA聚合酶原液。B：0.2 mmol/L IPTG诱导Kod DNA聚合酶表达。1：Kod DNA聚合酶粗提液（CSKod）;2：超声破碎后离心的残渣重悬液（CMKod）;3：透析后经储存缓冲液1∶1稀释后的Kod DNA聚合酶原液

Fig.1 Effects of different concentrations of IPTG on the expressions of Kod DNA polymerases The effects of different concentrations of IPTG on the expressions of Kod DNA polymerase. M：Protein molecular weight standard. A：0.1 mmol/L IPTG induces the expression of Kod DNA polymerase. 1：The crude extract of Kod DNA polymerase（CSKod）;2：suspension of residue after sonication and centrifugation（CMKod）;3：Kod DNA polymerase stock solution diluted with storage buffer at 1∶1 after dialysis. B. 0.2 mmol/L IPTG induces the expression of Kod DNA polymerase. 1：The crude extract of Kod DNA polymerase（CSKod）;2：suspension of residue after sonication and centrifugation（CMKod）;3：Kod DNA polymerase stock solution diluted with storage buffer at 1∶1 after dialysis

图2 不同浓度咪唑对Kod DNA聚合酶纯化的影响含有不同浓度咪唑洗脱液对Kod DNA聚合酶纯化回收产量的影响。M：蛋白分子质量标准;1-3：500 mmol/L、200 mmol/L、100 mmol/L咪唑洗脱液;CK：Kod DNA聚合酶粗提液（CSKod）

Fig.2 Effects of different concentrations of imidazole on the purifications of Kod DNA polymerases The effect of different imidazole concentrations contained in elution buffer on the purification yield of Kod DNA polymerase. M：Protein molecule quality standard;1-3：500 mmol/L,200 mmol/L 100 mmol/L imidazole eluate;CK：the crude extracti of Kod DNA polymerase（CSKod）

图3 优化条件后Kod DNA聚合酶的制备效果优化诱导和提取条件后,Kod DNA聚合酶的制备效果。M：蛋白分子质量标准;CK：Kod DNA聚合酶粗提液（CSKod）;1-3：200 mmol/L咪唑洗脱液洗脱第1次,第2次和第3次收集的Kod DNA聚合酶;4-8：浓度为0.5 mg/mL、1 mg/mL、0.25 mg/mL、0.125 mg/mL、0.0625 mg/mL的牛血清蛋白（BSA）

Fig.3 Preparation effect of Kod DNA polymerase after comprehensive optimization conditions Kod preparation after comprehensive optimization induction and purification conditions. M：Protein molecule quality standard. CK：The crude extraction of Kod DNA polymerase（CSKod）;1-3：elution times using 200 mmol/L imidazole;4-8：Different concentrations of BSA（0.5 mg/mL,1 mg/mL,0.25 mg/mL,0.125 mg/mL,0.0625 mg/mL）

图4 Kod DNA聚合酶PCR反应体系中酶浓度及Mg2+浓度对目的基因扩增的影响 M：5 000 bp DNA分子质量标准;1-24：扩增长度约为3 194 bp的AtVSR6片段;1、7、13、19：使用市售商品化GO Tag高保真DNA聚合酶扩增结果;2-6、8-12、20-24：分别使用浓度为0.061 25 μg/µL、0.125 μg/µL、0.25 μg/µL、0.5 μg/µL、1 μg/µL的Kod DNA聚合酶扩增结果;Mg2+1、Mg2+2、Mg2+3、Mg2+4：分别使用浓度为1.5 mmol/L、1.75 mmol/L、2.0 mmol/L和2.25 mmol/L

Fig. 4 Effects of enzyme content and Mg2+ concentration on target gene amplification in Kod DNA polymerase reaction system M：DL5000 DNA marker;1-24：amplification of AtVSR6 fragments with a length of about 3 194 bp;1,7,13,19：amplification product with commercial GO Tag high-fidelity DNA polymerase;2-6,8-12,20-24：amplified results using different concentrations of Kod DNA polymerase（0.061 25 μg/µL,0.125 μg/µL,0.25 μg/µL,0.5μg/µL,and 1μg/µL）;Mg2+1. Mg2+2,Mg2+3,Mg2+4：different concentrations of Mg2+（1.5 mmol/L,1.75 mmol/L,2.0 mmol/L and 2.25 mmol/L）

图5 Kod DNA聚合酶的保真性验证 A：M：5000 bp DNA分子质量标准;1-4：扩增片段长度为3 194 bp的AtVSR6片段;CK+：阳性对照;CK-：阴性对照。B,C：拟南芥AtVSR6基因测序结果与NCBI数据库中AtVSR6基因序列的比对结果

Fig.5 High-fidelity verification of Kod DNA polymerases A：M：DL5000 DNA marker;1-4：amplification of AtVSR6 fragments with a length of about 3 194 bp;CK+：positive control;CK-：negative control. B,C：The BLAST result between sequencing result of AtVSR6 gene in A. thaliana and AtVSR6 gene sequence in NCBI database

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