چكيده لاتين
Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase exhibiting template-independent polymerization activity, with significant relevance in research, industrial, and clinical applications. Consequently, the development of efficient and cost-effective purification strategies for this enzyme holds substantial practical value. Among the available methods, nickel-sepharose affinity chromatography is widely employed, enabling specific, single-step purification. However, this approach presents notable drawbacks, including high cost, the requirement for specialized equipment, and pronounced limitations in industrial scalability. In contrast, the polyethylene glycol (PEG)-based three-phase partitioning (TPP) system offers a simple, low-cost, and rapid alternative that has demonstrated scalability for numerous proteins. Nonetheless, TPP is limited by lower specificity and potential interference of phase components with enzymatic activity assays.
The present study aimed to compare the efficiency of nickel-sepharose affinity chromatography and the PEG-based TPP system for the purification of recombinant TdT expressed in Escherichia coli. SDS-PAGE analysis revealed that both methods yielded a protein band corresponding to the expected molecular weight of 45 kDa. Quantitative analysis indicated that the TPP system, employing 10% (w/v) PEG4000 and 80% (w/v) ammonium sulfate at pH 7.0, produced a higher total protein yield (2.53 ± 0.09 mg) and greater protein recovery efficiency (59.78 ± 2.17%) compared with affinity chromatography, which recovered 1.67 ± 0.16 mg of protein with a 39.23 ± 3.94% recovery rate. Conversely, affinity chromatography achieved markedly superior final purity (53.09 ± 0.37%) relative to TPP (14.98 ± 0.92%).
Given the polymerizable nature of TdT, its activity must be assessed using specialized assays such as Urea-PAGE. Samples purified via nickel-sepharose affinity chromatography can be directly evaluated without interference. In contrast, TPP-purified samples contain phase components—particularly high salt concentrations—that interfere with enzymatic activity assays. Therefore, desalting procedures, such as dialysis, ultrafiltration, buffer exchange, or gel filtration chromatography, are required prior to activity assessment.
Overall, this study demonstrates that TPP represents a scalable, economical, and rapid approach for the recovery of recombinant TdT, offering a viable alternative for enzyme purification processes where industrial-scale application is desired.
