I am expressing codon optimized fungal protein in E.coli. The protein has transit peptide in it, but in both the cases (with or without transit peptide) protein aggregated in inclusion bodies. I have kit that can be used for inclusion body protein extraction in its native form. But will there be any activity difference of protein express with / without transit peptide?
Hi there,
There is no golden rule concerning the possible effect of the presence of transit peptide on biological activity of the protein.
The role of a transit peptide is to direct a protein towards a secretory pathway, and since many fungal enzymes work on extracellular substrates it would be necessary have one of those.
Present at the N-terminus of an enzyme, the transit peptide may or may not get modified upon completion of secretion, which would also partially indicate if the modification was necessary for a fully functional enzyme.
In some cases, protein will not fold properly unless the leader peptide is cleaved.
I would substitute the intrinsic leader peptide with the OmpA or similar, so it would be
recognised by E. coli machinery. Alternatively, you could start the ORF directly from the
cleavage site.
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