Attempting to improve a target protein's solubility in recombinant expression. Currently His6 tagged and experiencing both low yield (inclusion bodies), and poorly soluble product (difficulty concentrating, precipitates easily).
Attempting to troubleshoot now by testing different expression and purification conditions before I try something more drastic like a fusion.
increasing the histidine residues in his tag does not improve solubility. it will for sure increase binding efficiency and will also tightly bind to chelating columns. this allows purification under stringent conditions. For a 10 x his tag you can wash at 100mM imidazole in your buffer and you can elute at 400mM Imidazole.
To increase solubility you might have to change the tag to MBP or GST. They will / might certainly increase solubility although there is always the risk that the protein might crash out if you cleave the tags. You might also want to try out inducing under different temperatures and in different expression hosts.
there is no straight path to proteins...you have to keep trying...
Good luck
Unlikely.
Add fusion tags that are known to increase solubility and folding, GST, SUMO etc. and stick a His tag on them for easy purification.
Good luck, Kirk.
If you don't want to try to add a fusion tag yet you could try decreasing the temperature just before induction from 37 oC down to 18-22 oC as well as decreasing the concentration of IPTG to 0.1-0.5 mM.
You'll probably have to do several trials to find the optimal conditions. What worked for us was a combination of the two.
Thanks, folks. Your answers were as I suspected. Will be trying the low temp expression next, before trying fusions. Best to all.
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