GST fusion protein extraction - (Oct/16/2005 )
Hi friends:
I need you help anxiously. I am using bacteria BL 21 to do GST pulldown. For the induction, there is no problem. I can get satisfied level of GST fusion protein. However, after havesting the bacteria and doing lysate, for some fusion protein, there was only a small part of GST fusion protein in soluble fraction. Most of the GST fusion protein is in insoluble fraction. My protocol for the lysis buffer of bacteria is : 1XPBS(cold) with protease inhibitor: Aptotinin, Leupeptin,Pepstain A. PMSF, Lysozyme, Triton X-100. In some protocol, I found someone use French press. Whether the method is really better than others?
hi,
Try to use lysis and washing buffer which contain urea.
good luck!
Use different conditions for induction. i think u r getting too late for protein. so try to get the samples from 15 min as it is forming inclusion bodies.. may be this will work if not try at room temp(21-22) for some more period of induction and see what happens. how much is mol wt of ur protein and which vector u r using?
Qiagen has some suggestions for purifying proteins that remain insoluble
try tween-20 instead of triton-x
add solubilization reagents such as: up to 20 mM beta-ME, up to 2 M NaCl, or a little Mg2++ to add some stability
(this is in some information on troubleshooting purfication of tagged proteins from E coli)
another possibility is to elute with a pH step-gradient
good luck
I think your problem is not on the lysis but on the production of soluble material. All proteins behave different when overexpressed.
Try different induction condictions (concentration of IPTG, temperatures, etc.). If it doesn't work you may try to change the expression vector. If you don't need the protein in native form, you can try expressing it with a His tag and just denature everything with urea and purify with a metal based resin (Ni or Co)...
Hope it helps
Try to use lysis and washing buffer which contain urea.
good luck!
Hi,
I have a problem with my protein. My protein is insoluble and my expressio levels are not satisfied. I'm trying different condition for induction, but my main problem is that the protein is in insoluble fraction.Can you give me a good concentration of urea for my lysis and washing buffer?Sorry for my english and because this is my first time in a bioforum..Thanks