title: Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria.
abstract: Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.
authors.affiliation: Life Sciences Division, United States Surgical Corporation, North Haven, Connecticut 06473, USA.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Buechter
authors.forename: David N
authors.initials: DN
authors.lastname: Paolella
authors.forename: Bridget S
authors.initials: BS
authors.lastname: Leslie
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Brown
authors.forename: Karen A
authors.initials: KA
authors.lastname: Mehos
authors.forename: Elliott A
authors.initials: EA
authors.lastname: Gruskin
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003062:Codon; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006909:Hydroxyproline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D024042:Collagen Type I; D011994:Recombinant Proteins; D011392:Proline; D005982:Glutathione Transferase; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; D006909:Hydroxyproline