title: Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins.
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.
authors.affiliation: Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Khattabi
authors.forename: Maarten L
authors.initials: ML
authors.lastname: van Roosmalen
authors.forename: Dennis
authors.initials: D
authors.lastname: Jager
authors.forename: Heidi
authors.initials: H
authors.lastname: Metselaar
authors.forename: Hjalmar
authors.initials: H
authors.lastname: Permentier
authors.forename: Kees
authors.initials: K
authors.lastname: Leenhouts
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D013294:Lactococcus lactis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*; D000097797:Glyphosate
chemical_list: D001426:Bacterial Proteins; D005998:Glycine; D011994:Recombinant Proteins; D014364:Tryptophan; D000097797:Glyphosate; C004182:5-methyltryptophan