Abstract
For development of a novel method in protein engineering, we studied the use of nonprotein amino acids, namely, amino acids other than the 20 protein-constituting amino acids. We succeeded in in vivo production of human epidermal growth factor (hEGF) substituted with norleucine, an analog of methionine. In the present study we attempted production of hEGF substituted with p-fluorophenylalanine residue (pFPhe), although hEGF does not have Phe residues. We prepared two genes of mutant hEGF; Tyr22 or Tyr29 was replaced by Phe by oligonucleotide-directed mutagenesis. To enhance incorporation of p-fluorophenyl-alanine, these mutant hEGF genes were introduced into an Escherichia coli strain auxotrophic for phenylalanine. The optimum concentration of p-fluorophenylalanine for production of pFPhe-substituted hEGFs was 50 μg/ml. By gel filtration chromatography, and reverse-phase and ion-exchange high performance liquid chromatography, we succeeded in purification of pFPhe-substituted hEGFs. Fluorine substitution of the aromatic ring of each Phe residue probably induces a conformational change of hEGF.
A variety of analogs of protein-constituting amino acids have been used for studies on protein synthesis, post-translational protein modification, and conformation — function correlations of peptides. We attempted production of proteins substituted with nonprotein amino acids (alloproteins) [1] and further development of novel protein engineering taking advantage of unnatural side chains of nonprotein amino acids. We succeeded in the preparation of human epidermal growth factor (hEGF1) substituted with norleucine (Nle) [1]. Preparation of Nle-substituted proteins has also been reported by other groups [2–4].
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© 1990 ESCOM Science Publishers B.V.
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Koide, H. et al. (1990). Biosynthesis of epidermal growth factor having nonprotein amino acid residues by Escherichia coli. In: Lubec, G., Rosenthal, G.A. (eds) Amino Acids. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-2262-7_23
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DOI: https://doi.org/10.1007/978-94-011-2262-7_23
Publisher Name: Springer, Dordrecht
Print ISBN: 978-90-72199-04-1
Online ISBN: 978-94-011-2262-7
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