Intein-mediated biotinylation of proteins and its application in protein microarray

Abstract

Protein microarray is the next grand challenge after the completion of the human genome project. A key factor critical to the success of protein array technology is the immobilization strategies. Herein, we describe a novel approach using intein-mediated expression system to generate biotinylated proteins suitable for highly stable, yet site-specific immobilization onto avidin-functionalized glass slides. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin tag. Using the intein-mediated biotinylation strategy, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput fashion. The biotin-tagged proteins were subsequently immobilized onto avidin-functionalized surfaces for applications such as protein microarray and surface plasmon resonance (SPR) analysis. In addition, we also demonstrated that intein-mediated protein biotinylation proceed within both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system.