title: Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation.
abstract: Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts (positions 1--51, 54--99, and 102--127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.
authors.affiliation: Department of Biochemistry/Chemistry, University of Western Australia, Nedlands Western Australia 6907, Australia.
authors.forename: J A
authors.initials: JA
authors.lastname: Wilce
authors.forename: S G
authors.initials: SG
authors.lastname: Love
authors.forename: S J
authors.initials: SJ
authors.lastname: Richardson
authors.forename: P F
authors.initials: PF
authors.lastname: Alewood
authors.forename: D J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011228:Prealbumin / Q000032:analysis / Q000138:chemical synthesis*; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D008024:Ligands; D011228:Prealbumin