Recombinant protein expression

Question

Hello! I'd like to ask a question about protein expression. My question is how I can remove N-terminal formil-methionine from E.coli recombinant protein if the next amino acid after methionie is phenylalanine. It is known that methionine aminopeptidases such as MAPs from Pyrococcus furiosus are depent on a penultimate residue of substrate and they do not react with Cys, Asp, Asn, Leu, Ile, Gln, Glu, His, Met, Phe, Lys, Tyr, Trp, Arg amino acids. So, How i can deal with this problem. What type of enzyme will be applicable for M↓F removal ?

Answer 1

There is no cut and dry answer on this and is a problem that has been explored in the literature. There have been engineered MAPs reported that can cleave the n-term methionine with a bulky residue in the 2nd position. These however aren't perfect and can sometimes cleave the 2nd residue if the 3rd is sufficiently small. Perhaps an easier, more reliable approach would be to do one cycle of the Edman Degradation, in which the sample is treated with phenylisothiocyanate, cleaving the n-term amino acid. Historically, the Edman Degradation was used to (painstakingly) sequence protein sequences.