Versatile protein biotinylation strategies for potential high-throughput proteomics

J Am Chem Soc. 2004 Feb 4;126(4):1055-62. doi: 10.1021/ja037914g.


We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. 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. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation / methods*
  • Cell Line
  • Cell-Free System
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Protein Array Analysis
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism
  • Proteomics / methods*
  • Surface Plasmon Resonance


  • Fungal Proteins
  • Luminescent Proteins
  • Proteins
  • Green Fluorescent Proteins