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Affinity adsorption of plasmid DNA

机译：质粒DNA的亲和吸附

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The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 muL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.
机译：这项工作描述了蛋白质介导的质粒DNA双重功能亲和吸附的发展。质粒DNA的亲和配体包含融合蛋白，其中谷胱甘肽-S-转移酶（GST）作为与锌指蛋白的融合伴侣。蛋白质配体首先通过GST部分与共价固定在基础基质上的谷硫硫酮之间的亲和相互作用与吸附剂结合。然后通过锌指蛋白和插入DNA中的特定核苷酸序列实现质粒结合。在较低的负载量下，DNA与Fractogel，Sepharose和Streamline基质结合的吸附量为50μL，分别为0.0078 +/- 0.0013、0.0095 +/- 0.0016和0.0080 +/- 0.0006 mg。在较高的DNA攻击下，相应的量分别为0.0179 +/- 0.0043、0.0219 +/- 0.0035和0.0190 +/- 0.0041 mg。与这三种吸附剂结合的相对恒定的量表明，大的DNA分子无法利用位于内部孔中的可用锌指位点，并且结合主要是表面吸附现象。对于Fractogel吸附剂，锌指结合位点的利用率最高。用还原型谷胱甘肽洗脱所吸附的物质，并且DNA的洗脱效率在23％至27％之间。蛋白质洗脱曲线似乎与吸附曲线相匹配，结合的GST-锌指蛋白回收率明显更高。

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