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Removal of N‐terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase
Author(s) -
Liao YouDi,
Jeng JenChong,
Wang ChiuFeng,
Wang SuiChi,
Chang ShuTing
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04679104
Subject(s) - recombinant dna , biochemistry , methionine , escherichia coli , protein engineering , residue (chemistry) , chemistry , biology , enzyme , microbiology and biotechnology , amino acid , gene
Abstract The removal of N‐terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate‐binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens . The N‐terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto‐toxicity. On the basis of N‐terminal sequence information in the protein database, 85%–90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.