A novel system for stable, high-level expression from the T7 promoter.
BACKGROUND
The most widespread, efficient prokaryotic protein-producing system is one where the T7 phage polymerase recognizes the T7 phage promoter (T7 p/p system). Unfortunately, in this system, target protein expression gradually declines and is often undetectable following 3 to 5 subcultures. Although a number of studies have attempted to stabilize the expression levels of the T7 p/p system, none has resolved the problem adequately and thus precludes the use of this system for the production of recombinant proteins on a large scale.


RESULTS
We created an expression cassette enabling stable, high-level expression in the T7p/p system. The cassette was tested with two different vector backbones and two target proteins. In all experiments, the expression system using the new cassette exhibited high and stable protein expression levels when compared to the traditional system.


CONCLUSIONS
Herein, we describe a universal expression cassette that enables high-level, stable target protein expression in T7 RNA polymerase-based expression systems. We also present the successful use of this cassette as a novel expression platform and demonstrate its ability to overcome the main deficiency of the T7 p/p system. Thus, we provide a method for using the T7 p/p system on an industrial scale.
Department of Bioengineering, Institute of Biotechnology and Antibiotics, Staroscinska 5, Warsaw, 02-516, Poland. kesikm@iba.waw.pl
Malgorzata
M
Kesik-Brodacka
Agnieszka
A
Romanik
Diana
D
Mikiewicz-Sygula
Grazyna
G
Plucienniczak
Andrzej
A
Plucienniczak
D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases