how to express protein in soluble form - (Mar/08/2011 )
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...
first of all play around with the inducer concentrations ...try lower concentrations.
after that you can try even lower culture conditions ...e.g. 37°C until a certain OD and after induction culture them at 16°C 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
samita on Tue Mar 8 13:47:45 2011 said:
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...