Nanobody Libraries Using Protein Ligation

Introduction Nanobodies (Nb) are single‐domain antibodies derived from camelids and are one‐tenth the size of normal antibodies. Various methods have been developed to obtain potent Nb using phage display and yeast display, but the processes of introducing mutations and evaluating Nb mutants are separated, making Nb acquisition a relatively time‐consuming and costly process. Recently, in vivo continuous evolution, in which genes are diversified within an organism, has been focused on as a means to accelerate the improvement of protein function. In in vivo continuous evolution, mutagenesis enzymes accumulate mutations in the target gene, and the target gene is continuously diversified simply by culturing the organism. Although in vivo continuous evolution is useful, it is not compatible enough with display technologies. For example, in yeast display, Nb is expressed as a fusion protein with a scaffold protein to be immobilized on the yeast cell wall. If the scaffold protein is mutated, the display itself does not occur. In this study, we have developed a method for binding scaffold proteins expressed from different gene cassettes to Nb in yeast and presenting them on the yeast surface using SpyTag/SpyCatcher [1] protein ligation, which irreversibly forms isopeptide bonds. This method can be integrated with in vivo continuous evolution because Nb can be expressed separately from the scaffold proteins required for the surface display system, allowing selective directed evolution of Nb alone. Methods As a model, we generated a yeast strain which produced anti‐lysozyme Nb fused with SpyTag (ST, 16‐residue) and a 649‐stalk scaffold protein fused with an epitope tag and SpyCatcher (SC, 113‐residue). To investigate potential crosstalk between yeast cells, two yeast strains, which produced different Nbs and epitope tag, were co‐cultured and stained with anti‐epitope antibody and fluorescently labeled lysozyme for microscopic observation. To confirm that our display method can be used for library screening, we attempted to enrich the target yeast cells using a flow cytometer. We generated a mixture sample of yeast cells displaying anti‐lysozyme Nb or Nb that does not bind lysozyme. The sample was mixed with fluorescently labeled lysozyme, and fluorescence‐positive cells were sorted. Results and Discussion Yeast cells producing anti‐lysozyme Nb fused with SpyTag (ST) and 649‐stalk scaffold protein fused with an epitope tag and SpyCatcher (SC) were stained with anti‐HA antibody and fluorescently labeled lysozyme, and we confirmed that 93% of the yeast cells successfully displayed anti‐lysozyme Nb on the yeast cell surface. We observed no crosstalk between yeast cells. In the enrichment experiment, we successfully confirmed that the fluorescence‐positive cells were enriched to about 94% by using a flow cytometer. In conclusion, our technique is expected to be an efficient cell surface display platform which can be integrated with in vivo continuous evolution[2]. [1] Anthony. et al. PNAS , Dec 2019, 116, 26523. [2] Kajiwara et al. submitted.