Klymkowsky Lab On-line Methods
Induction and purification of proteins expressed in bacteria

Table of Methods updated - 20 May 2002

Plakoglobin in the epidermis


Most of our bacterial expression system is based on the pET plasmid from Studier’s lab. This plasmid contaion a T7 promoter driving expression.

To make protein, the plasmid is transformed into a cell line (BL21DE3) in which the the gene encoding the T7 RNA polymerase has been inserted into the cellular genome.

Some plasmids use a lac promoter to drive expression -- these plasmids can be used to express proteins in the DH5a bacterial strain.

Both the T7pol genein BL21s and the lac promoter in DH5a are (of course) under lac control. Addition of IPTG induces T7pol synthesis in BL21s and the transcription of all genes regulated by a lac promoter in DH5a.

It should be noted, however, that a number of the plasmid we have made seem to express the exogenous protein consitutatively -- we are not sure why.

SO - for each induction experiment, use the following controls
An E. coli strain that does not contain an expression plasmid
Your test strain, grown without IPTG
Your test strain, grown with IPTG

  1. Before you grow up a large scale prep. for protein purification (see below), and if you only need a little protein for gel analysis, start with 5ml overnight cultures grown without the inducer IPTG.
  2. The next morning, dilute 1ml of culture with 4 ml of fresh LB -- all culture to grow at 37°C for 30 minutes and then add induced (1mM IPTG) diluted from 100X stock.
  3. Let induction proceed from 2 to 4 hours.
  4. Recover bacteria by centrifugation -- discard supernatant and resuspend pellet in 100-200 µL of SDS-sample buffer + 5% ß-mercaptoethanol.
    You will need to shear resuspended sample by sonication to breakup the DNA.
  5. Incubate at 60°C for 5 minutes at then centrifuge at high speed for 5 minutes.
  6. Load supernatant on gel.

Table of Methods