Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor.

• DOI: 10.1073/pnas.85.17.6237
• Publication Date: 2006-05-31T10:41:25Z
• Authors: H Koide; S Yokoyama; G Kawai; J M Ha; T Oka; S Kawai; T Miyake; T Fuwa; T Miyazawa
• Keywords: Aminocaproates; 0303 health sciences; Epidermal Growth Factor; Recombinant Proteins; 3. Good health; Mice; 03 medical and health sciences; Genes; Aminocaproic Acid; Escherichia coli; Animals; Humans; Cloning, Molecular; Promoter Regions, Genetic; Cell Division; Cells, Cultured; Plasmids

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.