title: Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein.
abstract: The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.
authors.affiliation: Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia. julien.tailhades@florey.edu.au.
authors.forename: Julien
authors.initials: J
authors.lastname: Tailhades
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Ashish
authors.initials: A
authors.lastname: Sethi
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Petrie
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
authors.affiliation: Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia.|Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Bathgate
authors.affiliation: Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia. john.wade@florey.edu.au.|The School of Chemistry, The University of Melbourne, Victoria, 3010, Australia. john.wade@florey.edu.au.
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.affiliation: Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia. akhter.hossain@florey.edu.au.|The School of Chemistry, The University of Melbourne, Victoria, 3010, Australia. akhter.hossain@florey.edu.au.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Hossain
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D065096:Calcium Chelating Agents / Q000737:chemistry*; D004274:DNA, Recombinant / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008026:Ligation; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D065096:Calcium Chelating Agents; D004274:DNA, Recombinant; C417543:RXFP1 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D001224:Aspartic Acid; C075871:aspartimide
keywords: NMR spectroscopy; insulin-like peptide; native chemical ligation; peptides; relaxin