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An expression system for the efficient incorporation of an expanded set of tryptophan analogues

Petrovic, D. M., Leenhouts, K., van Roosmalen, M. L. & Broos, J., May-2013, In : Amino Acids. 44, 5, p. 1329-1336 8 p.

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DOI

  • Dejan M. Petrovic
  • Kees Leenhouts
  • Maarten L. van Roosmalen
  • Jaap Broos

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (> 89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi-pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.

Original languageEnglish
Pages (from-to)1329-1336
Number of pages8
JournalAmino Acids
Volume44
Issue number5
Publication statusPublished - May-2013

    Keywords

  • Lactococcus lactis, Trp auxotroph, Tryptophanyl-tRNA synthetase, Bromotryptophan, Chlorotryptophan, Difluorotryptophan, SITE-SPECIFIC INCORPORATION, AMINO-ACID REPERTOIRE, LACTOCOCCUS-LACTIS, IN-VIVO, BIOSYNTHETIC INCORPORATION, RECOMBINANT PROTEINS, ESCHERICHIA-COLI, GENETIC-CODE, FLUORESCENCE SPECTROSCOPY, STREPTOCOCCUS-CREMORIS

ID: 5843443