title: Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase.
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.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115. ydliao@ibms.sinica.edu.tw
authors.forename: You-Di
authors.initials: YD
authors.lastname: Liao
authors.forename: Jen-Chong
authors.initials: JC
authors.lastname: Jeng
authors.forename: Chiu-Feng
authors.initials: CF
authors.lastname: Wang
authors.forename: Sui-Chi
authors.initials: SC
authors.lastname: Wang
authors.forename: Shu-Ting
authors.initials: ST
authors.lastname: Chang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008715:Methionine / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D011894:Rana pipiens / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D008715:Methionine; D012260:Ribonucleases; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; C058703:ranpirnase