auto induction system.pptVIP

Protein Production by Auto-Inductionin Inducible T7 Expression Systems  NMR Group National Laboratory of Macromolecules INSTITUTE OF BIOPYSICS,CASOctober 28, 2005 Evolution of a Widely Used and UsefulInducible T7 Expression System in E. coli Rationale for using elements of phage T7 Capabilities for high-level expression Reducing basal expression Preventing unintended induction Auto-induction for high yields and throughput Bacteriophage T7 Efficiently Takes Over the E. coli Cell Upon Infection T7 phage particle delivers T7 DNA to the E. coli cell Efficient mechanisms direct the resources of the cell toward the production of new T7 phage Can produce 250 new T7 phage particles in 13 min at 37?C Phage T7 生长周期：早期转录阶段和晚期转录阶段，早期转录使用E.coli的RNA聚合酶，晚期使用T7基因1编码的RNA聚合酶。  早期转录物包括一种激酶，使E.coli的RNA聚合酶失活，以及T7RNA聚合酶。 T7RNA聚合酶超强启动其他基因的转录。 Upon Infection, T7 Directs All Gene Expression in the Cell to its Own DNA T7 RNA polymerase is highly selective and processive T7 mRNAs are stable T7 mRNAs have strong upstream translation signals 乳糖操纵子（lac operon） 原核生物，某一生物学过程相关的蛋白质处于同一调控机制。 乳糖代谢：lactose permase，β-galactosidase，transacetylase Lac Operon： Inducible High-level Expression From the T7 Expression System Target mRNA can accumulate to levels that saturate ribosomes Target protein can accumulate to more than half of total cell protein Wide range of coding sequences expressed Broadening the Range and Convenience Original configuration of the T7 system has significant basal expression of target proteins Target proteins toxic or stressful to the host cell can destabilize or prevent establishment of expression plasmids Plasmids ------to love and to hate: a difficult choice for E.coli Two strategies to reduce basal expression Inhibit T7 RNA polymerase with T7 lysozyme Block target promoter with lac repressor Problems from Unintended Induction T7lac promoter can give very low basal expression, but Grossman et al. (Gene 209: 95-103 (1998)) found spontaneous induction near sat