Versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions

Anal Chem. 2014 Sep 2;86(17):8535-40. doi: 10.1021/ac501601g. Epub 2014 Aug 11.


Immobilization of a protein in a functionally active form and correct orientation for high-throughput analysis is crucial for surface-based protein-molecular interaction studies and should aid progress in associated nanotechnologies. Here, we present a general method for controlled and oriented immobilization of proteins by a puromycin-linker for cDNA display technology. The utility and potential of this method was demonstrated by examining the interaction between the B domain of protein A and immunoglobulin G (IgG) by surface plasmon resonance. This study revealed that the mRNA fragment of the mRNA-protein fusion (i.e., mRNA display) interferes with the interaction between the protein (B domain) and its target molecule (IgG). This results in a reduction of the apparent affinity by ~10-fold. This method is expected to find wide appeal in the fields of surface-based studies of protein-protein interactions, drug screening, and single molecule analysis that require only a small amount of protein sample.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / chemistry*
  • Biotin / metabolism
  • Biotinylation
  • Cell-Free System
  • Immobilized Proteins / chemistry
  • Immobilized Proteins / metabolism
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / metabolism
  • Protein Biosynthesis
  • Protein Interaction Domains and Motifs
  • Protein Structure, Tertiary
  • Puromycin / chemistry*
  • Quartz Crystal Microbalance Techniques
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • Staphylococcal Protein A / chemistry*
  • Staphylococcal Protein A / metabolism
  • Surface Plasmon Resonance*


  • Immobilized Proteins
  • Immunoglobulin G
  • RNA, Messenger
  • Staphylococcal Protein A
  • Puromycin
  • Biotin