Applying the Alkaline Lysis Method for rProtein A Extraction from E. coli Bacteria: A Simple Approach for Large Protein Sample Extraction

Introduction: The increasing demand for monoclonal antibodies in medicine requires scalable and efficient production methods. Because of its strong affinity for immunoglobulins, Staphylococcal protein A is a key tool for antibody purification. Conventional methods of extracting recombinant protein A (rProtein A), such as sonication, are costly, inefficient at high volumes, and difficult to scale up. Due to the inherent stability of rProtein A in alkaline pH, this study used NaOH to extract rProtein A from E. coli.Materials and Methods: The protein was expressed in E. coli BL۲۱ (DE۳), and then cell disruption was achieved through sonication and alkaline lysis. Ni-NTA affinity chromatography was employed to purify the isolated proteins. An ELISA-based assay evaluated the functionality and binding affinity of the purified rProtein A by determining the equilibrium dissociation constant (Kd).Results: Alkaline lysis proved more effective, releasing nearly ۹۸% of rProtein A from the cell pellet compared to ۴۰% with sonication. Additionally, the functionality of rProtein A remained intact, exhibiting excellent antibody-binding affinity with a Kd of ۶.۹۵ nM, slightly better than the ۷.۰۴ nM Kd of the sonication-produced protein.Conclusions: Overall, alkaline lysis is a reliable, cost-efficient, and scalable alternative to sonication for the primary recovery of rProtein A from bacterial cells.

کلیدواژه ها:

rProtein A ، Monoclonal antibody ، Sonication ، Alkaline Lysis ، Protein extraction