CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal; CDRSP-IPLEIRIA - Centre for Rapid and Sustainable Product Development, Instituto Politécnico de Leiria, Rua de Portugal - Zona Industrial, 2430-028 Marinha Grande, Portugal.
CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
J Chromatogr A. 2020 May 10;1618:460890. doi: 10.1016/j.chroma.2020.460890. Epub 2020 Jan 15.
p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.
p53 是一种肿瘤抑制基因，已被探索用于癌症基因治疗，作为常见治疗方法的一种替代方法。已经考虑使用质粒 DNA（pDNA）携带治疗基因，但为了引发更有效的基因表达和治疗作用，必须保持其超螺旋（sc）结构。已经成功应用基于氨基酸的亲和层析来纯化 sc pDNA，探索了不同的氨基酸和载体。在这些研究中，精氨酸对 sc pDNA 的识别选择性引人注目。然而，在精氨酸琼脂糖载体中发现了结合容量的一些限制，而在整体柱的情况下，一些堵塞和堵塞可能会限制连续运行。通过使用精氨酸修饰的大孔载体，可以利用配体的选择性结合载体提供的流动性能和结合容量。通过 SEM、EDX 和 FTIR 对精氨酸修饰的大孔载体进行了表征，以验证精氨酸的正确固定化，然后用于 pDNA 纯化。该载体对 sc p53-pDNA 的分离有效，通过略微调整实验条件，还实现了对不同大小质粒的纯化，从而实现了稳健性。关于精氨酸修饰的大孔载体的动态结合容量，与同源的精氨酸琼脂糖商业基质相比，pDNA 的结合容量提高了 50%以上，这表明在放大规模时具有潜在的经济可行性。
