Processing of N-terminal unnatural amino acids in recombinant human interferon-beta in Escherichia coli.
Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we report the effects of the penultimate residue (the residue after the initiator Met) on the processing of two unnatural amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), at the N terminus of recombinant human interferon-beta in E. coli. We have identified specific amino acids at the penultimate position that can be used to efficiently retain or remove N-terminal AHA or HPG. Retention of N-terminal AHA or HPG can be achieved by choosing amino acids with large side chains (such as Gln, Glu, and His) at the penultimate position, while Ala can be selected for the removal of N-terminal AHA or HPG. Incomplete processing of N-terminal AHA and HPG (in terms of both deformylation and cleavage) was observed with Gly or Ser at the penultimate position.
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D000596:Amino Acids; D007370:Interferon Type I; D011994:Recombinant Proteins; C506098:azidohomoalanine; D018698:Glutamic Acid; D012694:Serine; D006639:Histidine; D000409:Alanine; D005998:Glycine