PCold induction

The growth/induction conditions (culture medium, culture temperature, aeration, timing of induction, concentration of an inducer, and incubation time after induction) should be optimized for each target protein.

A general description of the protocol is shown below.

1) Insert the target gene fragment into the MCS of pCold DNA. Be sure that the sequence of the fragment is inserted in-frame with the fusion tag sequence.

2) Transform the E. coli host strain ( Nico21(DE3)pLysN) with this plasmid, and select the transformants on an agar plate containing K/C.

3) Inoculate LB medium containing 50  μg/ml of Kan, 35 ug/ml Chloramphenicol  with a few colonies , and incubate with shaking at 37℃.

4) When the OD600 of the culture reaches 0.4 - 0.8, quickly cool the culture to 15℃ in ice water, and let stand for 30 minutes.

5) Add IPTG at a final concentration of 0.1 - 1.0 mM, and continue the culture with shaking at 15℃ for 24 hours.

6) Collect the cells, and confirm the presence, amount, and solubility of the target protein using SDS-PAGE or an activity assay.

By optimizing the host strain, culture, and expression induction conditions (e.g., culture medium and temperature, degree of aeration and agitation, timing of induction, IPTG concentration, culture conditions after induction, etc.), it may be possible to increase the expression level and solubility of the target protein. Moreover, when the expressed protein is insoluble, the combined use of pCold DNA with Chaperone Plasmids may be effective.