Single-molecule detection of spontaneous native protein ligationView all posters
National Chiao Tung University, Taiwan
Direct amide formation between carboxylic acid and amine under physiological condition helps to build multiple-function protein assembly with native activities. However, the formation of peptide bond (amide) through condensation of carboxylic acid and amine requires high temperature and pressure. At physiological condition the condensation may occur in the absence of toxic catalyst; however, extremely low yield is expected. We plan to fish out few successful products out of large excess of reactants using single-molecule sensing and catching platform. A protein transistor made of single antibody binding two gold nanoparticles was fabricated. The electrical conductance across antibody monitors the structure dynamics and also the physical states of antibody in real-time. Pepsin treatment cleaved off the Fc domain of antibody leaving two Fab’ fragments connected to each other. The binding of pepsin and the cleavage of Fc domain was visualized by the increase and decrease of electrical conductance. When Fc fragment was delivered using microfluidic channel to Fab’s, an increase of conductance was observed and stably maintained. It was assumed that the Fab’ fragments were capable of catching and ligating Fc fragment at physiological condition. The reconstructed antibody exhibited similar electric characteristics compared to intact antobody. Negative differential resistance (NDR) was observed with intact antibody. NDR disappeared when Fc domain was cleaved. However, when Fc domain was ligated back to Fab’s, NDR appeared, indicating the structural integrity was recovered. Several enzymes were successfully ligated to the Fab’s, including DNA polymerase I, phi 29 DNA polymerase, glucose oxidase, horse relish peroxidase, beta-galactosidase, and T4 DNA polymerase. In particular, these conjugated enzymes showed comparable native activity. Although the yield of carboxylic acid/amine condensation is extremely low at physiological condition, we were able to fish out spontaneous formation of peptide bond using the ultra-sensitive single-molecule detecting system.