I am trying to express and purify a protein
after doing all the steps and purify using Ni affinity column
but after doing the SDS PAGE I found that the amount of protein in the elute is very little while it is very lot in the insoluble part
how can I solve this problem?
if I add lysozyme to the lysis buffer, this will make the protein soluble??
If the bacterial cells were broken open correctly to release the protein, adding lysozyme will have no effect. If the cells were not broken, lysozyme might help with breaking them. This can be monitored with a microscope.
Many proteins are insoluble when overexpressed. It sometimes improves solubility to slow down the expression. One way to do this is to grow the bacteria at a lower temperature. They grow more slowly and express the protein more slowly, giving it time to fold properly instead of precipitating.
There are other techniques you can try, such as changing expression strains, coexpressing chaperones, or making a fusion protein with a soluble partner such as maltose binding protein.
If the bacterial cells were broken open correctly to release the protein, adding lysozyme will have no effect. If the cells were not broken, lysozyme might help with breaking them. This can be monitored with a microscope.
Many proteins are insoluble when overexpressed. It sometimes improves solubility to slow down the expression. One way to do this is to grow the bacteria at a lower temperature. They grow more slowly and express the protein more slowly, giving it time to fold properly instead of precipitating.
There are other techniques you can try, such as changing expression strains, coexpressing chaperones, or making a fusion protein with a soluble partner such as maltose binding protein.
The best way is to attach fusion tag like thioredoxin, MBP, GST with your protein .
We do this using suitable vector having these tags, along with this there should be clevage site for sumo or TEV(these are very specific proteases) protease to remove these tags during purification.
Lysozyme only help in breaking down the bacterial cells not in solubilization of protein.
If you are using bacteria you may vary the concentration of iptg and temperature. If synthesis of your protein is high and occurs fast there is no time for proper individual protein folding and you end up with useless protein aggregates
I would also like to mention, using 1.5% Sarkosyl in the Tris-lysis buffer and sonicate. After sonication add Triton-X-100 to a final concentration of 2% (BMB Reports).
I have tried this method and it does work. Hope it helps. Thanks.
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