So the story is that my protein is taged with MBP. I tried to purify it both on 5 ml and 1 ml columns but the result was bad! It had very dirty flowthrough and wash, and the elution was with a tiny peak with faint band on SDS-PAGE. When I moved to sephrose beads I had a huge amount of protein in the elution and very clean BUT with lower MW than expected! (my protein is around 76 KDa + 42 for maltose =118 KDa more or less but the band seen was about 68 KDa!)
Can anyone help me?
the MBP tag is on the N-terminal. and yes i could verify the construct via sequencing. I expressed by autoinduction. I should note that i tried with another MBP tag vectpr (pMALc2x which has factorX as protease) and when i purified i had the band of my protein at the proper size but i decided to change to another MBP tag vector that has precession protease but now am getting the band at MW lower than expected .
there is a conflict in aa sequence within your protein and the junction of the tag with protein leading to auto cleavage or the tagged protein and low MW.
low expression can be due to the very high MW of the protein in ecoli system.
even if ecoli makes this protein in lage qty, it will be inclusion body not the soluble protein.
try expressing your 76kd protein with his-tag. or try without any tags, and use combination of pH and Ion exchange chromatography to purify it.
alternatively you can use the dialysis (50Kd cutoff and 100kD) to get rid of low MW proteins to enrich your 76 KD protein.
Sharad i tried it with His-tag before but it was insoluble (band in the pellet and nearly nothing in elution) and besides it was so dirty but with MBP i had huge amounts and very clean i can send it to mass spec. to make sure what is it but that will take time and i wanna know why the bands are in a lower MW on gel.
Maybe there is some impurities in your extract making the protein runs into the gel faster. Why you don't try precipitate the extract with TCA or acetone and then ressuspend in some urea or thioreia.. Just maybe... I don't have big experience on it. But I hope you can find a solution soon.
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