High-Yield Production and Purification of Recombinant Glucagon in Escherichia coli Using TRX Tag

Bakherad, Hamid; Abedi, Alireza

doi:10.30491/jabr.2025.513077.1858

High-Yield Production and Purification of Recombinant Glucagon in Escherichia coli Using TRX Tag

Document Type : Original Article

Authors

Hamid Bakherad ¹^{, 2}

Alireza Abedi ¹

¹ Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

² Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

10.30491/jabr.2025.513077.1858

Abstract

Introduction: Glucagon is a 29-amino acid peptide hormone with a molecular weight of 3,485 Da. It plays a critical role in increasing blood glucose levels, reducing involuntary gastrointestinal (GI) motility, and facilitating digestion. Intravenous administration of glucagon is widely utilized for the treatment of severe hypoglycemia in both pediatric and adult diabetic patients. Furthermore, glucagon is employed as a diagnostic agent in radiological procedures to temporarily inhibit GI motility in adult patients. In this study, we aimed to produce and purify glucagon using the TRX tag in E. coli BL21 (DE3).
Materials and Methods: In this study, the glucagon gene, fused with His and TRX tags, was cloned. The recombinant plasmid was subsequently introduced into E. coli BL21 (DE3) cells, and recombinant protein expression was analyzed following bacterial culture in Luria-Bertani (LB) medium. Following protein expression, an initial purification was conducted through several inclusion body (IB) wash steps, effectively eliminating most protein impurities. In the subsequent step, a Ni-NTA column was employed to achieve further purification, yielding a protein of high purity.
Results: The expression and purification procedures were optimized, resulting in the glucagon yield from this study using a shake flask (batch system) of approximately 16 mg/L. To facilitate the separation of the TRX tag from glucagon, enterokinase was employed as a cleavage enzyme.
Conclusions: The results indicated that the construct designed to produce glucagon had an acceptable production rate (16 mg/L), as well as enterokinase exhibited non-specific cleavage of the recombinant construct, rendering it unsuitable for this purpose. Therefore, TEV protease should be used as an alternative enzyme for effective tag removal in protein structure.

Keywords

E. coli

Glucagon

Recombinant Production

TRX Tag