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Published January 17, 2008 | Supplemental Material
Journal Article Open

Processing of N-Terminal Unnatural Amino Acids in Recombinant Human Interferon-β in Escherichia coli

Abstract

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-β 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.

Additional Information

c2008 Wiley-VCH Verlag. Issue published online: 17 JAN 2008. Article first published online: 20 DEC 2007. Manuscript Received: 10 JUL 2007. We would like to thank Inchan Kwon, Tom Graddis, Kurt Shanebeck, Scott Bloom, and J. Myron Crawford for their advice and assistance. Research on UAAs at Caltech is supported by NIH Grant GM62523.

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