CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
J Chromatogr A. 2013 Sep 13;1307:91-8. doi: 10.1016/j.chroma.2013.07.078. Epub 2013 Jul 26.
Affinity chromatography represents a sole technique in purification of different biomolecules. The specific recognition between affinity ligands and target biomolecules has a major role in the specificity of the process. Therefore, choosing the right ligand is a crucial step for the development of a successful purification system. This work describes the application of the DNA intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as a chromatographic affinity ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA). The support was prepared by coupling DAPP onto an epoxy-activated Sepharose matrix, using mild conditions and resulting in a ligand density of 0.15mmolDAPP/g derivatized Sepharose. The characterization of DAPP-Sepharose support in terms of dynamic binding capacity was achieved by studying the effect of plasmid DNA concentration and flow rate on pDNA adsorption. The maximum capacity value of 336.75μgpDNA/mL gel was obtained at 1mL/min with a pDNA concentration of 150μg/mL. Moreover, the values did not vary significantly with the variation of flow rate. In addition, the DAPP-Sepharose showed a high affinity towards pDNA as quantified by the respective dissociation constant (Kd=2.29±0.195×10(-7)M). The support was also tested for the purification of two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05kbp and 12.361kbp, respectively) from clarified Escherichia coli lysate solutions. Total retention of all lysate components was achieved without any added salt to the eluent buffer. The selective elution of impurities and supercoiled pDNA was accomplished simply by the addition of small amounts of salt to the buffer solution. The yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ was 94%, with all host impurity levels in accordance with the requirements established by the regulatory agencies.
亲和层析是一种纯化不同生物分子的单一技术。亲和配体与靶生物分子之间的特异性识别在该过程的特异性中起着重要作用。因此，选择合适的配体是开发成功的纯化系统的关键步骤。本工作描述了 DNA 嵌入剂 3,8-二氨基-6-苯基菲啶（DAPP）作为色谱亲和配体在超螺旋质粒 DNA（pDNA）的特异性分离和纯化中的应用。该支持物是通过将 DAPP 偶联到环氧活化的 Sepharose 基质上制备的，使用温和的条件，导致衍生化 Sepharose 的配体密度为 0.15mmol DAPP/g。通过研究质粒 DNA 浓度和流速对 pDNA 吸附的影响，实现了 DAPP-Sepharose 支持物在动态结合能力方面的特性。在 1mL/min 时，pDNA 浓度为 150μg/mL 时，获得了 336.75μgpDNA/mL 凝胶的最大容量值。此外，该值随流速的变化没有明显变化。此外，DAPP-Sepharose 对 pDNA 表现出高亲和力，其解离常数（Kd=2.29±0.195×10(-7)M）也有所量化。该支持物还用于从澄清的大肠杆菌裂解物溶液中纯化两种具有不同大小的质粒分子（pVAX1-LacZ 和 pCAMBIA-1303，分别为 6.05kbp 和 12.361kbp）。通过向洗脱缓冲液中不添加盐，实现了所有裂解物成分的完全保留。通过向缓冲液溶液中添加少量盐，简单地实现了杂质和超螺旋 pDNA 的选择性洗脱。pCAMBIA-1303 的收率为 65%，pVAX1-LacZ 的收率为 94%，所有宿主杂质水平均符合监管机构规定的要求。
