Engineering a high‐throughput clone for industrial‐scale production of long‐acting GLP‐1 analogue with retained bio‐efficacy

Abstract Type 2 diabetes mellitus (T2DM) and obesity are critical global health issues with rising incidence rates. Glucagon‐like peptide‐1 (GLP‐1) analogues have emerged as effective treatments due to their ability to regulate blood glucose levels and gastric emptying through central nervous signals involving hypothalamic receptors, such as leptin. To address the short plasma half‐life of native GLP‐1, a C‐16 fatty acid was conjugated to lysine in the GLP‐1 analogue sequence to enhance its longevity. This study focuses on engineering a high‐throughput clone and evaluation of novel GLP‐1 analogues with improved bio‐efficacy and production yields. Five plasmid models were created using different N‐terminal fusion partners and assessed for hydrophobicity, instability index, and isoelectric point. Three optimal plasmid models were selected based on high‐valued hydrophobicity, solubility, and partial solubility. These plasmids were constructed with the pET24a vector, incorporating GLP‐1 with fusion tags via recombinant DNA technology and transformed into E. coli BL21 DE3 hosts. The proteins were purified through enzyme digestion and chromatography, resulting in a high‐yield peptide. The GLP‐1 peptide was conjugated with in‐house developed fatty acid compound n‐Palmitoyl glutamic acid (n‐PGA) and purified using C18 column chromatography, achieving a final product yield of 170–190 mg per liter of fermentation culture. Biological activity was confirmed by cyclic adenosine monophosphate (cAMP) generation and 3 T3 cell differentiation assays, showing a 1.5‐fold increase in mRNA gene expression with the clone having n‐terminal hydrophobic amino acids, thioredoxin‐modified tag, and enterokinase cleavage site, indicating high purity and biological potency of the GLP‐1 analogue.