Yeast (Saccharomyces cerevisiae) methionine aminopeptidase I: rapid purification and improved activity assay-猫眼课题宝

酵母（酿酒酵母）甲硫氨酸氨基肽酶 I：快速纯化和改进的活性测定

DOI：

10.1111/j.1470-8744.1999.tb00545.x

发表时间：

1999

期刊：

Biotechnology and Applied Biochemistry

影响因子：

2.8

通讯作者：

中科院分区：

工程技术4区

文献类型：

作者：

K. Walker;Elizabeth Yi;R. Bradshaw

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 (k cat 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 μM peptide in a 100 μl 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.