混合模式层析分离纯化超螺旋质粒DNA

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

针对细胞裂解液中的超螺旋质粒DNA(sc pDNA)的分离,以质粒pVAX1为典型对象、采用Capto PlasmidSelect作为混合模式层析介质,探讨了料液中主要成分sc pDNA、开环质粒DNA(oc pDNA)和RNA的吸附行为,优化了分离条件,实现了从成分较为复杂的料液中高效分离sc pDNA。考察了上述3种组分的静态吸附,发现在(NH_(4))_(2)SO_(4)浓度c(NH_(4))_(2)SO4为1.9~2.5 mol·L^(-1)时,sc pDNA均具有较高的吸附量,确定c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)的料液可直接上样,此时sc pDNA饱和吸附量为每克介质吸附3.3 mg。动态吸附实验发现,sc pDNA穿透略晚于oc pDNA,sc pDNA动态载量为每毫升介质负载2.00 mg,RNA吸附能力明显强于pDNA。进一步优化了洗脱、冲洗和上样量等分离条件,采用c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)上样、c(NH_(4))_(2)SO_(4)=1.9 mol·L^(-1)冲洗、(c(NH_(4))_(2)SO_(4)=1.7 mol·L^(-1))+(cNaCl=0.3 mol·L^(-1))洗脱,sc pDNA纯度可达83.9%、同质性高达95.8%、收率为80.6%。结果表明,混合模式层析对sc pDNA选择性好、处理量较大,具有良好的应用价值。

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)(NH_(4))_(2)SO_(4)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)(NH_(4))_(2)SO_(4) 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)(NH_(4))_(2)SO_(4) for washing,and 1.7 mol·L^(-1)(NH_(4))_(2)SO_(4)+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.