I am using Chaperone Competent Cell BL21 from Takara, pGro7/BL21 , but it is not clear on the protocol how and when I should induce the expression of the chaperones with L-arabinose. First time I tried, I got this SDS-page showing only the overexpression of chaperone of 60 KDa; and I added 2mg/mL Arabinose.
Does anyone have a protocol for protein expression using these cells? Or any suggestion?
Thanks!
Hi Irene Feliciotti
You may find the following pdf helpful.
https://www.takara.co.kr/file/manual/pdf/9120-9125_e.v1810Da.pdf
Hi,
I think you should use 0.01% of L-Arabinose and for more information about this topic you may refer this research article.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047061/
1. Prepare L-broth containing 20 μg/ml chloramphenicol and 50 - 100 μg/ml of
ampicilin for plasmid expression, and 0.5 - 4 mg/ml L-arabinose (For pGro7), L-arabinose alone is added.
＊ Please try with 0.5 mg/ml L-arabinose at first.
2. Culture the transformant retaining pCold I DNA and Chaperone plasmid with shaking 250 rpm/ 4 h/ 37℃.
3. When the OD600 of the culture solution reaches 0.6 (3.5 h) , leave at 15℃ for 30 min.
4. Add IPTG at the final concentration of 1
mM, then culture with shaking 250 rpm at
18℃ for 24 hours.
5. After completion of the culture, verify the expression and solubility of a target protein
by SDS-PAGE or an activity assay.
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