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

Journal Article · Thu Sep 01 00:00:00 EDT 1988 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.85.17.6237· OSTI ID:5718809

Koide, Hiroshi; Yokoyama, Shigeyuki; Kawai, Gota; Ha, Jongmyung; Oka, Takanori; Kawai, Shintaro; Miyake, Tetsuo; Fuwa, Toru; Miyazawa, Tatsuo ^[1]

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1. Univ. of Tokyo (Japan)

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, the authors 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, (Ahx{sup 21})hEGF, secreted into the periplasm were recovered. After treatment with H{sub 2}O{sub 2}, (Ahx{sup 21})-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 (Ahx{sup 21})hEGF. The three biological activities of (Ahx{sup 21})hEGF were the same as those of hEGF. From the successful production of (Ahx{sup 21})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.

• OSTI ID: 5718809
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 85:17; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ESCHERICHIA COLI
GENE REGULATION
GROWTH FACTORS
HEXANOIC ACID
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LEUCINE
LIPOTROPIC FACTORS
LIQUID COLUMN CHROMATOGRAPHY
METHIONINE
MICROORGANISMS
MITOGENS
MOLECULAR STRUCTURE
MONOCARBOXYLIC ACIDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PLASMIDS
PROTEINS
RADIOISOTOPES
SEPARATION PROCESSES
SYNTHESIS
UPTAKE