Novel NTA-Ni2+ Agarose-Based Microspheres: Structural Features and Chromatographic Capacity.

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Citation abstract

The design and optimization of immobilized metal affinity chromatography (IMAC) media are crucial to enhancing the purification efficiency of recombinant proteins. In this study, the agarose-based microspheres are prepared by using a three-factorial Box-Behnken design followed by NTA-Ni 2+ agarose-based microspheres (ABM) preparation by the "one-step" crosslinking of epichlorohydrin (ECH)-nitrilotriacetic acid (NTA) to efficiently couple the NTA ligand to the surface of the matrix. After preparation, various sophisticated techniques, including SEM, AFM, DSC, FTIR, and SDS-PAGE, were used to analyze the morphological structure, thermal stability, and chemical composition of NTA-Ni 2+ ABM. The optimal conditions are identified as an emulsifier PP concentration of 8.12 wt%, a stirring speed of 1624.46 rpm, and an oil-phase temperature of 53.86 °C, giving a span value (Y) of 0.50684. SEM, AFM, DSC, and FTIR results showed that the fabricated NTA-Ni 2+ ABM were structurally stable and had a uniform cross-linking network for up to 8 h of coupling reaction time. The performance results showed that the beads had a high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL), and SDS-PAGE results demonstrated the efficient purification ability for target proteins. These findings provide the theoretical basis and a practical solution for the rational design and application of IMAC medium.