Research on the Preparation and Purification of Kod DNA Polymerase

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

Abstract

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

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.

Figures/Tables 6

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

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

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）

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）

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）

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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