Incorporation of 2-Aminoisobutyric Acid into Protein Therapeutics for Avoidance of DPP-4 Cleavage

Abstract

Glucagon-like peptide-1 (GLP-1) receptor agonists represent promising therapeutic agents for type 2 diabetes, obesity, and cardiovascular disease. However, their clinical development has been severely hampered by rapid enzymatic degradation through dipeptidyl peptidase-4 (DPP-4), resulting in an extremely short circulating half-life. To overcome these limitations, we developed a strategy combining site-specific incorporation of 2-aminoisobutyric acid (Aib) for DPP-4 resistance and fusion of an Albumin Binding Domain (ABD) for extended circulation. The primary challenge lies in achieving high-fidelity Aib incorporation through genetic code expansion (GCE) systems while maintaining cost-effective recombinant production. This study systematically optimized two key parameters: (1) plasmid architecture controlling orthogonal tRNA expression, and (2) Aib substrate concentration. Screening of plasmid variants revealed significant differences in Aib incorporation fidelity between pEVOL and pALS systems, with the pALS architecture employing a stronger promoter demonstrating superior performance. Subsequent dose-response analysis of Aib substrate concentration in the GLP-1 analogue demonstrated concentration-dependent enhancement of incorporation efficiency with stable protein yields across all tested conditions. The optimal conditions were identified and applied to achieve high-purity Aib- incorporated GLP-1 analogue while maintaining robust protein expression. This systematic optimization provides a validated methodology for establishing high-fidelity genetic code expansion systems applicable to therapeutic protein production, potentially enabling cost-effective recombinant synthesis of improved GLP-1-based therapeutics with enhanced proteolytic stability and extended pharmacokinetic properties.