Separation and purification of supercoiled plasmid DNA by mixed-mode chromatography

被引：0

作者：

Zhang P. ^{[1
]}

Tan Y. ^{[1
]}

Sun Y. ^{[1
]}

Zhang Q. ^{[1
]}

Yao S. ^{[1
]}

Lin D. ^{[1
]}

机构：

[1] Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou

来源：

Gao Xiao Hua Xue Gong Cheng Xue Bao/Journal of Chemical Engineering of Chinese Universities | 2023年 / 37卷 / 05期

关键词：

adsorption; mixed-mode chromatography; nucleic acid separation; supercoiled plasmid DNA;

D O I：

10.3969/j.issn.1003-9015.2023.05.013

中图分类号：

学科分类号：

摘要：

The mixed-mode resin Capto PlasmidSelect was used to study adsorption behaviors of supercoiled plasmid DNA sc pDNA, open-circular plasmid (oc pDNA) and RNA in order to purify (sc pDNA) from cell lysate with pVAX1 plasmid as a studying object. The separation conditions were then optimize to efficiently purify sc pDNA from clarified lysate. Static adsorption of sc pDNA, oc pDNA and RNA was investigated, and the results show that sc pDNA had high adsorption capacity at 1.9-2.5 mol‧L-1 (NH4)2SO4 salt concentration. The saturated adsorption capacity of sc pDNA was 3.3 mg‧g-1 resin, and the feedstock containing 2.5 mol‧L-1 (NH4)2SO4 could be loaded directly. The dynamic binding experiments indicate that sc pDNA penetrated slightly later than oc pDNA, and the dynamic binding capacity of sc pDNA was 2.00 mg‧mL-1 resin. The adsorption of RNA was significantly stronger than that of plasmid DNA. The separation conditions including elution and washing conditions and loading volume were optimized. The best conditions are 2.5 mol‧L-1 (NH4)2SO4 for loading, 1.9 mol‧L-1 (NH4)2SO4 for washing, and 1.7 mol‧L-1 (NH4)2SO4 + 0.3 mol‧L-1 NaCl for elution. The purity of sc pDNA reached 83.9%, the yield was 80.6% and the homogeneity was as high as 95.8%. The results demonstrate that mixed-mode chromatography has good selectivity and productivity for supercoiled plasmids. © 2023 Zhejiang University. All rights reserved.

引用

页码：806 / 812

页数：6

共 22 条

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