Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda.
Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.
Menart
Viktor
V
Lek d.d., Recombinant Biotechnology Department, Recombinant Pharmaceuticals, Laboratory location: Hajdrihova 19, SI-1000 Ljubljana, Slovenia. viktor.menart@ki.si
Jevsevar
Simona
S
Vilar
Mateja
M
Trobis
Andreja
A
Pavko
Aleksander
A
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000821:virology*; D005798:Genes, Bacterial; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha