期刊
MICROBIAL CELL FACTORIES
卷 11, 期 -, 页码 -出版社
BMC
DOI: 10.1186/1475-2859-11-109
关键词
T7 expression; Expression cassette; Stable expression from T7 promoter; High expression from T7 promoter
资金
- European Community funds of the European Fund for Regional Development [POIG.01.01.02-00-007/08-00]
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 T7 p/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.
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