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Protein Science (2004), 13:1802-1810. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

You-Di Liao, Jen-Chong Jeng, Chiu-Feng Wang, Sui-Chi Wang and Shu-Ting Chang

Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115

(RECEIVED February 5, 2004; FINAL REVISION April 1, 2004; ACCEPTED April 1, 2004)



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.

Keywords: N-terminal Met; E. coli MetAP; substrate specificity; penultimate residue; ribonuclease

Reprint requests to: You-Di Liao, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115; e-mail: ydliao{at}ibms.sinica.edu.tw; fax: 886-2-27829142.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04679104.


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