Engineered GLP-1 Fusion Proteins for Albumin Binding

The amino acid sequence for the DARPin with a high affinity for human serum albumin was obtained from US Patent 2016/9458211 B1⁶² . The GLP-1 molecule, which originally consisted of 30 amino acids from human GLP-1 (7–36), was modified with the substitution of alanine (Aln) by glycine (Gly) at position 8 to prevent DPP-IV recognition and degradation and with glutamine (Gln) and aspartic acid (Asp) instead of lysin (Lys) at positions 26 and 34, respectively, to inhibit trypsin digestion. Based on previous studies, the N-terminal of GLP-1 is important for its biological function; therefore, we engineered the fusion constructs to free the N-terminal of GLP-1. The native or two modified GLP-1 (mGLP-1) with Ala8Gly substitution or Ala8Gly, Lys26Gln, and Lys34Asp substitutions were genetically fused to the N-terminus of HSA-binding DARPin through a rigid helical linker (EAAAK)₃ to create the fusion proteins, denoted as nGLP1-DARPin, mGLP1-DARPin-1, and mGLP1-DARPin-2, respectively. Rigid linkers are used when there is a requirement to preserve the spatial distance between the domains and their independent functions.