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

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 similar to 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.