title: Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.
abstract: Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Mantas
authors.initials: M
authors.lastname: Liutkus
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Fraser
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Karine
authors.initials: K
authors.lastname: Caron
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Dannon J
authors.initials: DJ
authors.lastname: Stigers
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. Chris.Easton@anu.edu.au.
authors.forename: Christopher J
authors.identifier: http://orcid.org/0000-0002-2263-1204
authors.initials: CJ
authors.lastname: Easton
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002116:Calcitonin / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D019886:Gastrin-Releasing Peptide / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D056690:Prolactin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D002352:Carrier Proteins; D010455:Peptides; D056690:Prolactin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D019886:Gastrin-Releasing Peptide; D002116:Calcitonin
keywords: amino acids; cell-free synthesis; cleavage reactions; lactamization; peptides; protein expression