title: An expression system for the efficient incorporation of an expanded set of tryptophan analogues.
abstract: 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.
authors: Laboratory of Biophysical Chemistry and GBB Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors: Dejan M
authors: DM
authors: Petrović
authors: Kees
authors: K
authors: Leenhouts
authors: Maarten L
authors: ML
authors: van Roosmalen
authors: Jaap
authors: J
authors: Broos
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013379:Substrate Specificity; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism*; D014369:Tryptophan-tRNA Ligase / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014364:Tryptophan; D014369:Tryptophan-tRNA Ligase