A method for rapid nanobody screening with no bias of the library diversity

Abstract

Nanobody refers to the variable domain of heavy-chain only antibodies. The distinctive advantages of nanobodies including small size, feasible expression in Escherichia coli (E. coli), and superior stability make them promising tools for applications in scientific research and therapies. So far, the screening and expression of nanobodies are mainly following similar methods used for conventional antibodies, suffering from amplification-caused losses of the diversity of libraries and requirements of subcloning of interests into the expression vector. Here, based on the unique properties of nanobodies, we developed a new high-throughput (>105) method to screen and express nanobodies simultaneously, simplifying the production of specific nanobodies significantly. Fusing pelB signal peptide on the N-terminus, functional nanobodies were secreted into the culture medium, facilitating the direct screening of nanobody libraries and the subsequent purification of the target nanobody. Nanobodies specifically binding with target proteins were enriched through cell splitting and regrown steps in a way following the Poisson distribution to ensure no positive clones were dismissed, while the population of positive clones increased by one order of magnitude upon each round of cell splitting. In the end, 5 different nanobodies against the death domain receptor 5 (DR5) and 5 different nanobodies against the Pyrococcus furiosus (Pfu) DNA polymerase were produced directly out of their immunized libraries respectively. Our approach offers an integrated strategy for rapid, and low-cost nanobody screening and expression with no loss of the library diversity, demonstrating general applicability in the routine production of monoclonal nanobodies for diverse biomedical applications.

An NMR-guided approach for identifying co-factors in boosting the Pfu DNA polymerase

“…This includes the identification of binding sites (hot spots) for specific substrates in the working solution and the exploration of changes of hot spots on both enzyme and its substrates. Here 1 H- 13 C HMQC spectra of Pfu polymerase were measured in the absence and presence of a substrate hairpin DNA to identify the hot spots, which were further confirmed by applying home screened hot-start nanobodies (24). By targeting these hotspots, we identified the chemical chaperone, L-arginine, and the heat shock protein, HSP20 from Thermococcus kodakaraensis engaged with Pfu polymerase to bolster its thermal resilience, processivity and enhance its activity in amplifying lengthy DNA fragments.…”

“…S1A), which were defined as potential DNA binding sites. As the chemical shift perturbation may arise from the binding site and the allosteric conformational changed region, we then employed two home-screened hot-start nanobodies (NB2 and NB4) (24) to further confirm the binding hot spots of the hairpin DNA on the Pfu polymerase. Adding unlabeled NB2 or NB4 nanobodies into the 13 C labeled Pfu polymerase resulted in substantial CSPs of NMR resonance of a set of peaks (Fig.…”