how to express protien in soluble form - protien (Apr/01/2008 )
dear all i m expression plant protien n BL21 DE3 in pET28a vector at 37 for cell growth and at 30 for IPTG 0.5mM Induction and the protein goes to inclussion bodies how to express in the soluble form
or how to purify the active protien
waiting for reply
tkr
or how to purify the active protien
waiting for reply
tkr
You can try induction at a lower temperature ie room temperature or try induction at different IPTG concentrations and also a reduced shaking speed. I optimised mine to induction at 1.5mM growth at room temp and shaking at 200rpm for 6 hours induction. shaking at 150 rpm gave me reduced yield but it would probably give me better if i had left it longer. you need to do a time-course and run on sds to determine the best yield at different conditions. I could not get my protein fully in soluble fraction but it was better than at 37C, and 250 rpm. Anyhows, the soluble fraction is not enough for me so i would purify it in the denatured form with urea.
induce at 15 degree o/n would be better, if it doesn't work, try the chaprone DNA by co-tansfection.
another way is to change you Ecoli to ActicExpress.
You might also try to make truncations of the gene. If there are nonessential regions at the N or C terminus, remove them. It is very difficult to dramatically change the solubility of a protein, without taking a big hit on yield. Re-folding sometimes works to get soluble proteins, but restoring enzymatic activity may be another matter.
I would try expression in insect cells, if that is available to you.
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