I am purifying a 8 KDa protein tagged with GST and cloned in pGEX 6P 2 vector. When I cleaved 8 KDa protein from GST by prescission protease I got very less amount of 8 KDa Protein compare to tagged GST protein. Cleavage reaction conditions are as follows- cleavage Buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0), Incubation time 4 Hours.
I am also attached gel photo for the same.
Please tell me why it happens?
How I will get more amount of 8 KDa protein?
I agree with M.A.Shah that 8kDA protein is small hence it can be stained by coomassie according to its molecular size. what I miss in you picture is, the fusion protein GST-8kDA (uncleaved protein), which means the protease digestion is highly efficient!, isnt it?
If you want to have more proteins, then you just start purifying from maxi cultures (or optimize the condition of protein production). To control your digestion reaction, always add equal amount of uncleaved protein(that are not treated with protease but kept in the same condition) on a lane parallel to the samples that are digested (at different timepoints/conditions) to make sure about the efficiency.
Perhaps after cleave the GST tag you lost part of teh protein as aggregates or unfolded protein. GST tag is used to stabilize and facilitate the folding of the protein when is expressed by the bacteria, but sometimes after cleave it the free protein can precipitate. Have you observed some precipitate?
There could be several explanations for this.
Do you have a sample that you ran before protease clevage? How is the sample after your 1st purification? Is there a mixture of just GST and GST+8kDa protein?
Your 8kDa protein may be unstable and precipitates/degrades or gets chewed up. Maybe GST keeps the 8Kda protein in solution and once you cleave the tag it is unstable. You can add a short solubility tag to that construct in that case.
@Tamjeed Saleh Yes before cleavage i got very sharp single band ~34KDa and there was no mixture of GST and PEST.
You seem to be getting good overexpression of the tagged protein. The intensity of protein band on SDS will depend on the binding of CBB dye. Now GST is a larger protein and will show more prominent band as compared to 8kda protein. In order to get more of the digest,you need to do more digestions and pool the purified protein. it also happens sometimes that the cleavage site is partially accessible to protease. In that case, people use a low concentration of denaturant like urea or GdnHCl to effect cleavage.
best wishes
I agree with Mohammad, according to your picture it seems that the digestion was not sufficient enough.
I agree with M.A.Shah that 8kDA protein is small hence it can be stained by coomassie according to its molecular size. what I miss in you picture is, the fusion protein GST-8kDA (uncleaved protein), which means the protease digestion is highly efficient!, isnt it?
If you want to have more proteins, then you just start purifying from maxi cultures (or optimize the condition of protein production). To control your digestion reaction, always add equal amount of uncleaved protein(that are not treated with protease but kept in the same condition) on a lane parallel to the samples that are digested (at different timepoints/conditions) to make sure about the efficiency.
I agree with Mohammad and Ananda. If the 8 kDa protein stains with CBB as efficiently as GST, you would still expect
Its not clear if your digestion is complete. So when you run the gel next time please include 2 lanes. 1st the undigested protein and 2nd the digested protein lane so that you can see a difference in a shift of the GST band that would suggest the complete digestion. Now that you are showing only the digested land it does not distinguish between the digested and undigested band of GST which would be just 8kDa difference. Similarly you cannot compare the protein bands on the Coomassie intensity since the binding of the dye to the large molecular wt protein would be more intense than the low molecular wt protein and hence would show a difference. Check the protein concentration by Bradford. There could be chances of precipitation of the 8kDa protein since it was been stabilized for bacterial expression by GST tag. Precipitation does not mean a visible ppt but could be aggregation too.
Hi Mohd
I am also using pGEX expression system for my protein. Instead of precission cut site, it carries thrombin cleavage site. I see a fine band, when I purified my protein using GST sepharose beads, using anti-GST antibody. But when I tried cleaving the tag off, didn't see anything in elutes. I was wondering what percentage gel do you use she you want to run 8Kda protein, what are the conditions you transfer your protein: wet/dry/semi-dry? Any help is appreciated.
@Rohini Sachdeva I am using 12% SDS PAGE for running my protein. I have also tried with TRICINE-SDS PAGE for the same. The transfer condition for my protein is wet.
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