I want to do 2,3-butanediol dehydrogenase(BDH) enzyme purification to confirm its activity for 2,3-butanediol. Before that, I need to confirm which N or C terminal tagging is better for enzyme purification.
I checked BDH's protein structure by AlphaFold2. However, I got Predicted Metrics, predicted Local Distance Difference Test (pLDDT), MSA Sequence Coverage, and Predicted Aligned Error (PAE).
But I couldn't judge which terminal tagging is better for BDH enzyme purification. May I ask what protein structure prediction is for? How could I know which terminal tagging is better from the results? Or another way to know.
I research some papers. Some of them mentioned that it was ok to use whichever N or C terminal tagging.
You can look at the published or predicted structure to choose which end of the protein to tag. You want to put the tag on the end at which it is least likely to interfere with the assembly or function of the protein. The position should be solvent-facing, not involved in protein-protein interactions (in the case of an oligomeric protein), and not near the active site.
Tags can also be made to be removable by proteolytic cleavage after purification. Some journals insist that functional information to be published about enzymes should be collected from enzymes without tags still attached.
Dear Wu Yuzheng
When tagging proteins with N-terminal or C-terminal tags, the choice between these options can significantly impact the protein's functionality, stability, and expression.
The choice between N-terminal and C-terminal tagging should be guided by the specific goals of your experiment, the role of the protein’s terminal regions in its function, and the potential impact on folding, stability, and interactions. In general, C-terminal tags are often preferred when less likely to interfere with protein function, while N-terminal tags might be used when the protein’s N-terminal region is less crucial or when specific advantages are needed.
Thanks & Regards,
Lenin Kumar B.
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