Recombinant protein production driven by the tryptophan promoter is tightly controlled in ICONE 200, a new genetically engineered E. coli mutant.
Batch processes for recombinant gene expression in prokaryotic systems should optimally comprise a growth phase with minimal promoter activity followed by a short phase favoring expression. The strong promoter of the tryptophan operon (Ptrp) gives high-level expression of recombinant proteins in E. coli. The inefficiency to control basal expression before induction is however a major obstacle towards the use of Ptrp, especially in the case of toxic proteins. To circumvent this problem, a novel E. coli strain has been generated. This mutant, named ICONE 200 (Improved Cell for Over and Non-leaky Expression), underwent replacement of tnaA, the tryptophanase encoding gene, with the trpR gene encoding the aporepressor of Ptrp. Detailed analysis of ICONE 200 showed that tryptophan, in addition to its natural role of Ptrp co-repressor, was able to induce trpR through the tryptophan-inducible tryptophanase promoter/operator. Consequently, Ptrp-dependent expression was efficiently repressed in the presence of tryptophan and was turned on, as in wild-type E. coli, as soon as tryptophan was exhausted from the medium. ICONE 200 has the capacity to express a wide range of proteins including toxic proteins such as HIV-1 protease and poliovirus 2B protein. ICONE 200 is a new host carrying stable, targeted, and marker-free genetic modifications and a candidate for large-scale applications.
Chevalet
L
L
Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint-Julien en Genevois, France. laurent.chevalet@pierre-fabre.com
Robert
A
A
Gueneau
F
F
Bonnefoy
J Y
JY
Nguyen
T
T
D001426:Bacterial Proteins; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D016333:HIV Protease / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D016333:HIV Protease; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014364:Tryptophan; D017361:Viral Nonstructural Proteins; C091441:2BC protein, Poliovirus; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins