Gene cloning and utility phophorylation assay of a protein-fused substrate for a highly sensitive detection of cdc2 protein kinase using a radioisotope detection technique for the development of a protein biochip

✍ Scribed by Kyong-Cheol Ko; Mi Hee Choi; Sang Hyun Park

• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: French
• Weight: 269 KB
• Volume: 52
• Category: Article
• ISSN: 0022-2135
• DOI: 10.1002/jlcr.1579

✦ Synopsis

Abstract

The prototype of the cdc2 protein kinase in mammalian cells regulates its entry into mitosis by phosphorylating a group of key proteins in the major cell cycle transitions. In this study, using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, a rumen bacteria, a Mep45‐fused substrate (PKTPKKAKKL‐Mep45, MFS‐cdc2) was cloned to detect the activity of cdc2 protein kinase. We report here on a strategy for the detection of a phosphorylation of a substrate catalyzed by cdc2 protein kinase by using a radioisotope detection technique. It is possible to constantly obtain a reasonable quantity of MFS‐cdc2 for the cdc2 protein kinase assay and its cost can be as low as a synthesized peptide. Results of the study indicate that the Mep45‐fused protein can be used effectively as a substrate for detecting the activity of cdc2 protein kinase and it can be used in developing a protein biochip for a high‐throughput screening and also for studying protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.