Existence of beta-methylnorleucine in recombinant hirudin produced by Escherichia coli.
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 beta-methylnorleucine (beta MeNle; (2S,3S)-2-amino-3-methylhexanoic acid). The modification pathway of beta MeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, beta MeNle 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 beta MeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.
Muramatsu
Ryo
R
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, 3-17-35 Niizo-Minami, Toda, Saitama 335-8502, Japan. muramatu@j-energy.co.jp
Negishi
Toru
T
Mimoto
Tsutomu
T
Miura
Akira
A
Misawa
Satoru
S
Hayashi
Hideya
H
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006629:Hirudins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D007532:Isoleucine / Q000378:metabolism; D008745:Methylation; D009646:Norleucine / Q000031:analogs & derivatives* / Q000032:analysis*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007532:Isoleucine; C032957:beta-methylnorleucine; D009646:Norleucine; D014357:Trypsin; D013917:Thrombin