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how to express protein in soluble form


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#1 samita

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Posted 08 March 2011 - 05:47 AM

I am expressing a mutant protein, wt protein is also going into pellet but significant amount of protein is in soluble form and I can purify as well. But the mutant one is just in the pellet and nothing in the soluble form. I am expresing at 37 but I will try to 30 C as well. I have check the expresson in different cells as well. in all its going into pellet. What strategies should be used else then a 30 C to get the protein in soluble form. BL21-Gold, JM-109, Plys strain I have used...

Edited by samita, 08 March 2011 - 05:49 AM.


#2 pDNA

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Posted 08 March 2011 - 07:18 AM

first of all play around with the inducer concentrations ...try lower concentrations.

after that you can try even lower culture conditions ...e.g. 37C until a certain OD and after induction culture them at 16C overnight.

There are also strategies to add e.g. ethanol (1%) to the culture to increase the expression of chaperones.

If u use the T7 system and nothing works i would try to switch to a weaker promoter like lac and tac.

Regards,
p

I am expressing a mutant protein, wt protein is also going into pellet but significant amount of protein is in soluble form and I can purify as well. But the mutant one is just in the pellet and nothing in the soluble form. I am expresing at 37 but I will try to 30 C as well. I have check the expresson in different cells as well. in all its going into pellet. What strategies should be used else then a 30 C to get the protein in soluble form. BL21-Gold, JM-109, Plys strain I have used...






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