I am trying to purify a protein that is stably expressed in E.coli and I can stably purify a good amount. However, when I N-terminal tagged it with GST for another experiment and purified, I got good soluble yield. I pooled the protein into a centricon spin column and put it at 4degoC to concentrate it the next day.
When I resumed concentrating the next day, problem occurred! The protein had aggregated and am currently trying to salvage my protein from this mess! Please help.
Thank You.
Hi there,
First question : is the untagged protein stable after lysis and concentration? If yes the problem is linked to the presence of GST... Your GST fusion is unstable in the lysate and/or sensitive to concentration... Try to adapt the lysate buffer composition in order to limit this aggregation. I guess the GST tag has been chosen for purification purpose so what you can do is to affinity purify your fusion protein from the lysate without having to concentrate the sample : concentration will occur on the glutathion column... An other solution is to consider an other tag like MBP which is known to improve protein solubility. Hope it helps...
Hi Vinod, can you run samples of the aggregated protein on SDS PAGE +/- BME with boiling.?
If the aggregate is due to misfolded proteins then you should be able to see some resolution of different species. in the SDS lane.
If the aggregate is due to glutathione reducing free -SH groups in your expressed protein, causing them to form internal S-S bonds, then you will see resolution of the aggregate in the BME +SDS lane
problem could be two fold. Your protein doesn't like being at 4C or when it is concentrated it crashes out of solution (both have happened to me). If it is a concentration effect, try altering the salt concentration. If its cold temps don't put it in the fridge.
Thank you all for the answers.
@ Ross Thomson, I guess the problem is as you described, concentrating crashes the protein out of the solution! So, you recommend increasing the salt concentration? Currently it is sitting at very low concentration (uM range) in a MOPS buffer with 10mM NaCl.
I take it you are purifying gst tagged protein in PBS? If so then up the salt to 150mM and see if it stays in solution. The other issue could be the PI of the protein is close to pH of the buffer.
@ Ross Thomson, thank you for the reply. Not in PBS. I had to affinity purify it first since the protein has an N term His tag. So, I used 50mM Tris at pH 8, 500mM NaCl, 30mM Imidazole for the lysis, the protein was quite soluble in this (even though the pI of the protein is 8.06). So, since the protein was still soluble, I used the NiNTA column and eluted my protein. Following this I pooled the like fractions and tried to change the buffer to 5mM MOPS, pH 6.5, 10mM NaCl. During the process of concentrating the protein in order to change the buffer that my protein crashed out of the solution. I have lost considerable amount of my protein. But after removing the aggregated part, now at this low concentration (uM range), my protein seems quite stable. But I guess I will increase the NaCl concentration to 100mM to test its stability upon increasing the concentration.
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