Hi there,

To get a clearer idea on what is going on during the purification process you should perform MS fingerprint on the band to get the exact identity of the cleaved/degraded protein.

You can do partial trypsin digestion on crude lysate to see if you get the same band as in the purified sample.

It is most likely the degradation product and you may need to be more careful with your cooling setup during purification.

1. Work in the cold room if you can.

2. Keep solutions, and fractions being collected in water + ice mixture, putting solutions on the ice itself does not work very well. All tubes/bottles with solutions and fractions should be submerged in the ice water mix.

Proteolysis of fusion proteins between the polypeptide tag (GST, in this case) and the target protein is quite common, particularly if the linker segment is exposed in the 3-D structure of the fusion polypeptide (which by itself is a desirable feature if the tag is to be removed subsequently by some specifc protease like Tev protease, factor Xa etc.). There are many proteases in E. coli extracts with low sequence specificity that could effect the cleavage of the linker. Commercial companies sell various cocktails of protease inhibitors, which people routinely add to crude extracts in the hope to reduce the problem, albeit with moderate success much of the time. I concur with @Amit Pandey's suggestion: keep everything cold and and process your extract quickly to get rid of proteases as soon as possible

Sometimes, GST is cleaved during bacterial growth. To know that, spin your cells and resuspend them in SDS-PAGE sample buffer, sonicate the sample (you probably need a 100-200µl volume unless you have a very small sonication probe) to break the chromosomal DNA which would give (smears), boil immediately and load your samples on a gel. If you still have the cleaved GST band, no matter how fast and cold you work, you'll always get it. The solution is then to shorten the linker between the GST and your protein by mutagenesis or any modern cloning method. You can sometimes even remove the first residues of your protein and fuse the GST directly to the 2nd, 3d (and so on) residue (assuming GST is fused to the N-terminus). Make a few constructs like that and choose the less cleaved version.

Olivier Danot cleavage is only occurring after purification. The cleaved band is not present in the bacterial lysate. I am working in a cold room and with ice water this week and will see if that helps.

Working in a cold-room and keeping lysis, wash, and elution buffers on ice-water yielded same outcome = degraded fusion protein at the approximate size of linker+tag.

Hi,

Degradation of GST-tag fusion proteins is highly likely to occur, especially there is a restriction site between the target protein and the tag. You need to speed up the purification process and keep it at a low temperature as much as possible. In addition, it may be that the properties of the target protein are unstable, or the current conditions of expression and purification cannot guarantee that your target protein is in a stable state. Therefore, it is recommended that you try more constructs and expression systems.

As I said, you can still try to shorten the linker.