I tried to express a protein in the bacteria system (BL21 (DE3)). This expressed protein in any temperature and condition shows a bond lower than expectation on SDS-PAGE (theoretical MW:66284 Da). Can I attribute this difference to its high isoelectric point (PI:9.16)? I should mention this protein is a single polypeptide of a trimeric protein.
Total number of negatively charged residues (Asp + Glu): 65 Total number of positively charged residues (Arg + Lys): 77
Hello Parastou,
At first glance, it would seem that the heavy band of ~60 kDa is a proteolytic product of the 66 kDa band present in relatively higher amounts in the induced than in the noninduced samples. This would be consistent with the higher intensity of the 66 kDa band in the soluble fraction obtained from the strain expressing a chaperone. What is a bit surprising is the very few bands that you see in the soluble fraction. This looks rather like a pattern for the extracellular proteins secreted into the medium and not like a soluble cytoplasmic extract.
Assuming that your protein bears an affinity tag, the next step would be to try an affinity purification on the material that you get from the soluble fraction of the chaperone-bearing strain and see what you get on SDS-PAGE.
Dear Parastou,
SDS-PAGE provides an apparent molecular weight, with in most cases is quite close to the actual molecular weight but there are quite a number of cases in which this is not true. So I wouldn't be worried too much about this little deviation of the apparent mol weight from the expected one. Your explanation with the high pI may be true for your protein, but on the other hand, an even more basic protein like hen egg lysozyme (pI= 11,2) runs at the expected molecule weight in SDS-PAGE. There are other and better biochemical and biophysical methods to determine the precise molecular weight of a protein, if you are interested in this. SDS-PAGE is a good first bet, but not more. By the way, to catch a trimer by SDS-PAGE, you would have to chemically cross-link the polypeptide chains before SDS-PAGE. There are only few oligomers that survive boiling SDS, some actually do when you don't boil, like heterotrimeric complexes of MHC II-alpha and -beta and a suitable peptide like HEL46-61!
Best regards and good luck,
Robert
Dear Parastou,
I think Pierre Béguin has raised a very good point worth considering as well!
Best regards,
Robert
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