Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli

• Published in: Journal of biotechnology 2002-02, Vol.93 (2), p.131-142
• Main Authors: Muramatsu, Ryo, Negishi, Toru, Mimoto, Tsutomu, Miura, Akira, Misawa, Satoru, Hayashi, Hideya
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; b-methylnorleucine; Biological and medical sciences; Biotechnology; Chromatography, High Pressure Liquid; CX-397; Edman degradation; Escherichia coli; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Hirudin; Hirudins - analysis; Hirudins - chemistry; Hirudins - genetics; Isoleucine - metabolism; Methylation; Norleucine - analogs & derivatives; Norleucine - analysis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Mapping; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Thrombin; Thrombin - antagonists & inhibitors; thrombin inhibitors; Trypsin - metabolism; β-Methylnorleucine
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(01)00396-0

A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l −1) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as β-methylnorleucine (βMeNle; (2 S,3 S)-2-amino-3-methylhexanoic acid). The modification pathway of βMeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, βMeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of βMeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.