I am working on expressing a protein in E. coli BL21 and initially used the pET-21a vector, but the protein was completely insoluble and ended up in the pellet. To improve solubility, I switched to pET-22b with an N-terminal SUMO tag. After optimization, I observed a slight shift of the protein into the supernatant under the following conditions:
- Expression: 18°C, 16 hours, 0.5 mM IPTG
- Elution: NPI-250 buffer
- Yield: ~1 µg in 2400 µL
However, after dialysis, I found that no protein remained. I am trying to determine the cause of this loss and how to prevent it. Could this be due to precipitation, instability, or issues with buffer composition? I would appreciate any insights on optimizing the process to retain the protein after dialysis.
Thank you in advance.
It seems likely that precipitation or instability during dialysis is the primary cause of your protein loss. Optimizing the buffer conditions, maintaining a low temperature during dialysis 4°C, and considering the addition of stabilizers should help you retain more of your protein post-dialysis.
Nicolas Poirier, Thank you for helping me out. Changing the buffer composition did help me.
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