Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

Biochem J. 2008 Jan 1;409(1):193-8. doi: 10.1042/BJ20070909.

Abstract

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Biochemistry / methods*
  • Cloning, Molecular
  • Escherichia coli / metabolism
  • Gene Deletion
  • Gene Expression Regulation, Bacterial
  • Glycine / analogs & derivatives
  • Glycine / chemistry
  • Glyphosate
  • Lactococcus lactis / metabolism*
  • Recombinant Proteins / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Tryptophan / analogs & derivatives*
  • Tryptophan / chemistry*

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • 5-methyltryptophan
  • Tryptophan
  • Glycine