I'm trying to insert a linker between two proteins that is almost 100 amino acids in length, does anyone have any experience designing such a linker? I plan to order a gene block from IDT and insert it into the plasmid, (current the linker is only 36 bp when I need it to be almost 400bp in length).
Is there a reason for the linker to be so long? Linkers can be very short while allowing flexibility (e.g. hinge region in the FCB2 yeast protein). A 100 amino acids sounds like a lot, not to mention the possible interactions it can form with either protein (aggregation, loss of activity, etc...). I would try a 25 AA linker or a different linker sequence of the same length. If the linker is up to ~80bp, the DNA synthesis costs are minimal and several linkers can be ordered.
While planning a linker, consider the following:
- determine the general linker structure (loop, α helix, and so on). There are general amino acid motifs that form determined structures and can help you decide on a sequence. If the two proteins interact, a flexible loop might be necessary
- If the two proteins do need to interact, plan the linker length according to the distance between the two active domains (perhaps this is the reason for your desired linker length)
- After deciding on a sequence, check the PI of your new protein just in case
- Try this site - https://www.ibi.vu.nl/programs/linkerdbwww/ (based on doi: 10.1093/protein/13.5.309. )
- Badges
- Science topic