Methionine aminopeptidase (MetAP), in two isoenzymic forms, is responsible for hydrolysis of the initiator methionine residues from the majority of newly synthesized proteins. It is an essential gene product and is ubiquitously found in archaea, eubacteria, and lower and higher eukaryotes. MetAP also has a potentially important role in the production of recombinant proteins, since failure to correctly remove initiator methionine residues can result in a product that is inactive or immunogenic. A rapid two-step purification of recombinant yeast (Saccharomyces cerevisiae) MetAP I that produces a product that is more than 95% pure and maintains a high level of activity (kcat 320-1556 min-1) has been developed. In addition, a versatile, accurate and sensitive assay for MetAP activity is presented. In contrast with previous methods, which usually use short tripeptides and require a post-reaction derivatization step, this assay uses peptides ranging in size from four to eight residues and utilizes UV detection of a tryptophan residue at the C-terminus. As little as 1 pmol of yeast MetAP I can be detected, with 5 microM peptide in a 100 microl reaction. The combination of a rapid purification protocol and a significantly improved activity assay will allow for a detailed examination of MetAP structure-function relationships and may lead to improved enzyme reagents for use in recombinant-protein production.

• PMID: 10075912
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Walker KW, Yi E, Bradshaw RA

Primary Lit For

MAP1

Additional Lit For

Review For