Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins.
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.
El Khattabi
Mohamed
M
Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
van Roosmalen
Maarten L
ML
Jager
Dennis
D
Metselaar
Heidi
H
Permentier
Hjalmar
H
Leenhouts
Kees
K
Broos
Jaap
J
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
D001426:Bacterial Proteins; D005998:Glycine; D011994:Recombinant Proteins; D014364:Tryptophan; D000097797:Glyphosate; C004182:5-methyltryptophan