2 replies to this topic

[snig]

Hi all,

I'm having some trouble with solubility of TEV protease - I'm expressing a His-Tagged TEV protease in about 1 L of BL21(DE3) E. coli at 30 C using 1 mM IPTG as the inducer for about 4 hours.  However, in all samples I've looked at, all the protease is insoluble.  I was wondering if anyone had any tricks, outside of lowering the IPTG concentration or the temperature, on solublization.

Thanks a bunch!

[sudhakar mutyala]

Hi

Try first those lowering IPTG and temp....... what ever first then findout . if still ur prtoein is in insoluble then probably ur expression levels are very high or ur Protein it self is a probelm as some of the proteins never form soluble fractions because of their high molecular weight or hydrophobiciy or protein nature.


Regards
Sudhakar
I'm having some trouble with solubility of TEV protease - I'm expressing a His-Tagged TEV protease in about 1 L of BL21(DE3) E. coli at 30 C using 1 mM IPTG as the inducer for about 4 hours.  However, in all samples I've looked at, all the protease is insoluble.  I was wondering if anyone had any tricks, outside of lowering the IPTG concentration or the temperature, on solublization.

Thanks a bunch!
[/quote]

[dr.Phd]

snig, on May 7 2009, 06:37 PM, said:

Hi all,

I'm having some trouble with solubility of TEV protease - I'm expressing a His-Tagged TEV protease in about 1 L of BL21(DE3) E. coli at 30 C using 1 mM IPTG as the inducer for about 4 hours.  However, in all samples I've looked at, all the protease is insoluble.  I was wondering if anyone had any tricks, outside of lowering the IPTG concentration or the temperature, on solublization.

Thanks a bunch!

Try the following book chapter on TEV expression. your answer might lie in expression system. changing the construct to incorporate a MBP tag might help in preventing aggregation.

Expression and Purification of Soluble His6-Tagged TEV Protease
Joseph E. Tropea, Scott Cherry, and David S. Waugh
In       Sharon A. Doyle (ed.), Methods in Molecular Biology: High Throughput Protein Expression and Purification, vol. 498

Cheers!