Development of a high-yield recombinant clone for the enhanced production of a long-acting Glucagon like peptide-1 analogue with improved biological efficacy

被引:0
|
作者
Reddy, J. Praveen Kumar [1 ,2 ]
Tummuru, Murali [1 ]
Ramkumar, Kunka Mohanram [2 ]
机构
[1] Virchow Biotech Private Ltd, Hyderabad 500043, Telangana, India
[2] SRM Inst Sci & Technol, Sch Bioengn, Dept Biotechnol, Kattankulathur 603203, Tamil Nadu, India
关键词
Glucagon-like peptide-1 (GLP-1); Hypoglycaemia; Cyclic Adenosine monophosphate (cAMP); C-16 fatty acid compound; Enterokinase; ESCHERICHIA-COLI; GLP-1; RECEPTOR; LIRAGLUTIDE; EXPRESSION; CAMP; PURIFICATION; PROTEINS; CLEAVAGE; STRATEGY; PHARMACOKINETICS;
D O I
10.1016/j.procbio.2024.09.001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Glucagon-like peptide-1 (GLP-1) analogues have demonstrated greater efficacy and safety in treating type 2 diabetes mellitus (T2DM) due to their strong ability to regulate hypoglycemia. The short plasma half-life of native GLP-1 has prompted the development of novel strategies, such as the conjugation of a C-16 fatty acid to lysine in the GLP-1 analogue sequence, to enhance its longevity. With the escalating burden of T2DM, there is a pressing need for the development of innovative GLP-1 analogues with enhanced efficacy and increased production capacities. In this study, we engineered three recombinant DNA-based clones by incorporating distinct upstream fusion tags, enzyme cleavage sites, and charge-based additional amino acid sequences. These constructed plasmids were subsequently transformed into E. coli BL21 DE3 hosts to enhance solubility and streamline downstream protein purification and were evaluated for yield, purity, and biological effectiveness. A high-yield clone was selected, and the peptide was purified using column chromatography, yielding 150-180 mg per Liter of fermentation culture. The purified C-16 fatty acid-conjugated GLP-1 analogue peptide was analysed for its biological activity, particularly cAMP generation in pancreatic (3-cells. Results revealed a concentration of 10.58 pmol/mL of generated cAMP and an average 1.5-fold up-regulation of genes in the cAMP downstream pathway compared to the innovator standard. Based on these findings, we conclude that the developed clone, featuring the enterokinase cleavage site with a sequence of modified TrxA tag with five additional hydrophobic amino acids, not only enhances yield but also preserves the biological efficacy of the final product.
引用
收藏
页码:278 / 286
页数:9
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