With in vitro transcription translation I want to produce fusion protein with eGFP. I use the following linker: (..DELYK) - GSGSSGSGGS - (KDASA...). Is it possible that Gly-Ser linker is cleaved by proteases or that the ribosome falls off bevore the whole fusion protein is produced? I have problems with different lenghts of the produced proteins and I don't know why.
Thank you for your help.
With the information that you provide one can only speculate. Is it incomplete synthesis or proteolysis that you observe? If the later, then maybe the system you use (insect lysate I suppose) contains some protease that targets your fusion?
Why do you think the linker is the source of the problem? Have you tried to transfect your construct into any mammalian cells? Did you check the sequence of your construct? Did you optimize the codon usage for the system of interest?
Make sure that you are not using identical codon within SG linker. This would deplete certain tRNA much faster than others and it is often the reason of reduced and/or prematurely aborted translation.
Could be that K/HDEL is an ER import signal at the carboxy terminus and cleaved or processed? Could be a reason for the different lenghts of the produced protein.
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