Editing PCold induction

The growth/induction conditions (culture medium, culture temperature, aeration, timing of&nbsp;induction, concentration of an inducer, and incubation time after induction) should be optimized&nbsp;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&nbsp;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&nbsp;on an agar plate containing K/C.

3) Inoculate LB medium containing 50 &nbsp;μg/ml of Kan, 35 ug/ml Chloramphenicol&nbsp; 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&nbsp;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&nbsp;at 15℃ for 24 hours.

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

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