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OVEREXPRESSION > PRIT SYSTEMS

> Purification of Fusion Protein on IgG Sepharose 6 FF

> Induction of Temperature Sensitive Lambda Repressor

 

Induction of Temperature Sensitive Lambda Repressor

  1. Transform your plasmid DNA into N4830-1 cells (these cells have the gene for the temp. sensitive repressor CI857)

    NOTE: Your protein should be cloned into a vector which has the lambda promoter- the PRIT vectors have this.

    Transform also a control plasmid that has the lambda promoter and the fusion carrier but not a fusion partener. Incubate at 30ºC (non-permissive) overnight.

    Pick one colony into 2mL LB AMP and grow to saturation O/N at 30ºC. Shift temperature to 42ºC (permissive temp) by mixing with an equal volume of LB AMP pre-heated to 54ºC. Incubate at 42ºC for 80 minutes.
  2. Determine if your protein was overexpressed.