We are looking to produce recombinant trypsin without the activation peptide (which is 24aa). However, in order for the cells to produce the protein, the translation must start at Met, so there must be an initiation codon. However, how can we ensure that the initiation codon (Met) will be removed if the whole signal peptide is missing? We want to remove this codon and obtain the active protein intact from the 25th aa and beyond. How can we do that? Our cell lines are from Oncorhynchus Mykiss and Rattus Norvegicus. We know that Met aminopeptidase removes the Met from the start of the protein but we aren't sure the cells can perform this procedure.
It all depends on the identity of the amino acid at position P1' (i.e. following the amino-terminal Met). There is data in the literature regarding the substrate specificity of human Met amino-peptidases (for instance, PMID 20521764) but you'd have to look for papers on their rat and trout homologs.
If you must guarantee the identity of the N-terminal aminoacid and don't mind about labor or cost, the easiest way is to use an N-terminal SUMO fusion and remove the tag in vitro using the corresponding protease.
Hope it helps!
Alejandro, thanks a lot for your answer. The problem is that we don't want to interfere by using SUMO because we need to produce a system for the direct and large scale production of digestive enzymes on those cell lines. We have read that if after methionine the aa is small, then the METAP removes methionine succesfully and does not if the next aa is big. However, we don't want to add any aas after the methionine because it will alter our enzyme's sequence.. We thought about cloning our protein's sequence with the signal peptide in order for it to be removed by the cell's own METAP. Does that sound better to you?
Oh I see. So, your plan is to produce the active enzyme intracellularly? I mean, if you were to try to secrete it to the culture medium you wouldn't need to worry about METAP, since you'd have to use a signal peptide anyway.
Are you sure activated trypsin isn't toxic when produced intracellularly?
We decided that we will.include the signal and activation peptides and produce trypsinogen ... Then we will perform a treatment with enterokinase and get trypsin... So no worries about met.
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