title: Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein.
abstract: Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Yuelin
authors.initials: Y
authors.lastname: Kong
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Tong
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, United States.
authors.forename: Mingming
authors.initials: M
authors.lastname: Gao
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, PR China. Electronic address: xdgao@cpu.edu.cn.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, PR China. Electronic address: wbyao@cpu.edu.cn.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D052242:Adiponectin / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D004041:Dietary Fats / Q000378:metabolism; D004926:Escherichia coli; D017930:Genes, Reporter; D005813:Genes, Synthetic; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use; D000067757:Glucagon-Like Peptide-1 Receptor / Q000187:drug effects; D006943:Hyperglycemia / Q000097:blood / Q000188:drug therapy; D006949:Hyperlipidemias / Q000097:blood / Q000188:drug therapy; D007004:Hypoglycemic Agents / Q000494:pharmacology / Q000627:therapeutic use; D008055:Lipids / Q000097:blood; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D000069463:Olive Oil / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D052242:Adiponectin; D004041:Dietary Fats; D000067757:Glucagon-Like Peptide-1 Receptor; D007004:Hypoglycemic Agents; D008055:Lipids; D000069463:Olive Oil; D010455:Peptides; D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1; D055372:AMP-Activated Protein Kinases
keywords: Adiponectin; Diabetes; GLP-1; Linker engineering