I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX vector.I do the expression (induction 24 degree 16 hrs) and purification from Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity chromatography purified protein (cleaved protein after removal of tag) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern being retained after FPLC. Intensity of both the bands remain same even after one or two week of storage at 4 degree. Peptide mass fingerprinting (after trypsin digestion) suggest both are my proteins except minor difference in some peptide peaks. Is it because of rare codon/degradation of protein of interest or any other possibilities? Can I use this mixure for crystallization?
With Regards Vikrant
