The trigger factor (TF) fused with my target protein( a enzyme), will the TF affect the function of the protein? such as the affinity of substrate? Becuase of the big molecular mass of the TF. I found that the recombinant enzyme, fused with TF, showed higher Km, which means the lower affinity of substrate. now, I am writing the MS, can it be said that the lower affinity may be caused by fused-TF?
Thanks!
Dear Sir. Concerning your issue about the effect of the fused trigger factor TF tag on the function of the protein. Recombinant expression of eukaryotic proteins in bacteria often results in misfolding and aggregation. The ribosome-binding Trigger factor (TF) is the first molecular chaperone that interacts with nascent polypeptide chains in bacteria. Here we show that mutant TF lacking the PPIase domain (TFNC) is more efficient than wild-type TF in enhancing the folding yield of multi-domain proteins such as firefly luciferase. We find that TFNC has a shorter residence time on nascent chains, thus facilitating co-translational folding. By delaying folding relative to translation, the PPIase domain may increase the propensity of misfolding for certain eukaryotic proteins that rely on a mechanism of co-translational, domain-wise folding. I think the following below attached files may help you in your analysis.
Thanks
If the wild-type protein and the fusion protein have different Kms for the substrate under otherwise identical conditions (preferably measured at the same time), then it is logical to conclude that the presence of the fusion protein is affecting the Km. However, you need to be sure that the Km difference is statistically significant, so measure it at least 3 times and perform a t-test.
I know of at least one journal that says in the instructions to authors that it does not accept publication of kinetic measurements from proteins with uncleaved tags.
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