Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase.
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.
Liao
You-Di
YD
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115. ydliao@ibms.sinica.edu.tw
Jeng
Jen-Chong
JC
Wang
Chiu-Feng
CF
Wang
Sui-Chi
SC
Chang
Shu-Ting
ST
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
D000596:Amino Acids; D011994:Recombinant Proteins; D008715:Methionine; D012260:Ribonucleases; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; C058703:ranpirnase