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

Kooshki, Hamid; Mohammadi, Mozafar; Ebadi, Mehdi; Hashemzadeh, Mohammad Sadegh

doi:10.30491/jabr.2025.555733.1935

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

Document Type : Short Communication

Authors

Hamid Kooshki ¹

Mozafar Mohammadi ²

Mehdi Ebadi ²

Mohammad Sadegh Hashemzadeh ¹

¹ Nanobiotechnology Research Center, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

² Applied Biotechnology Research Center, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

10.30491/jabr.2025.555733.1935

Abstract

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 BL21 (DE3), 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 98% of rProtein A from the cell pellet compared to 40% with sonication. Additionally, the functionality of rProtein A remained intact, exhibiting excellent antibody-binding affinity with a Kd of 6.95 nM, slightly better than the 7.04 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.

Keywords

rProtein A

Monoclonal Antibody

Sonication

Alkaline Lysis

Protein Extraction