In vivo protein or peptide ligation has been achieved using SpyTag/Sp圜atcher-related technologies 22, 23. Protein/peptide ligation may enable the integration of YSD and in vivo continuous evolution. Therefore, a method to display the produced POI in trans to the anchor proteins is needed for integrating YSD and in vivo continuous evolution. This method has achieved an on-target mutation rate in the order of 10 −3 bp −1 and has succeeded in generating mitogen-activated protein kinase 1 that is resistant to both selumetinib and trametinib in just 2 weeks 18.Īlthough in vivo continuous evolution is useful, it requires some modification for its integration with YSD because mutagenesis can also introduce detrimental nonsense and missense mutations into the gene encoding the anchor protein, thus inhibiting the cell surface display of the POI. For example, one of the in vivo continuous evolution methods uses a cytidine deaminase fused to a T7 RNA polymerase introduces mutations into only a target gene under T7 promoter 18. In this strategy, a mutagenic enzyme which specifically recognizes a target gene continuously diversifies it by simply culturing the host cells (Supplementary Information Fig. S1).Ĭonversely, in vivo continuous evolution, in which target genes are diversified within an organism, has garnered much attention as a way to accelerate protein engineering 15– 21. Conventional directed evolution is a time-consuming process involving labor-intensitive rounds of in vitro gene diversification, the transformation of randomised genes into host cells, the selection of improved genes and the extraction of improved gene sequences from host cells (Supplementary Information Fig. This figure was created using Illustrator CS2 ( ).ĭirected evolution is widely used to improve the properties of a POI 10– 14. negative control Lys hen egg-white lysozyme RFI relative fluorescence intensity. secretion signal Nb nanobody GPI glycosylphosphatidylinositol attachment signal Ter terminator ST SpyTag SC Sp圜atcher N.C. The data shown are representative of two independent experiments. The ratio of yeast cells is shown in the upper right (UR) corner of each graph. Each sample was stained with mouse anti-HA tag antibody, AF488-conjugated anti-mouse antibody and AF647-labelled lysozyme. Details of plasmids construction for each strain are shown in Supplementary Information Fig. (c) The confirmation of the cell surface display of anti-lysozyme nanobodies based on the in vivo and in vitro SpyTag/Sp圜atcher-based protein ligation. The reactive Lys31 in Sp圜atcher and Asp117 in SpyTag are shown in the inset.
The asterisk denotes the intracellular isopeptide bond formation between a Sp圜atcher and a SpyTag. The schematic of (a) a conventional and (b) the SpyTag/Sp圜atcher-based yeast cell surface display.
Yeast cell surface display of the nanobodies generated by SpyTag/Sp圜atcher-based protein ligation. These results suggested that our system demonstrates comparable performance with conventional YSD methods therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting.
A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a Sp圜atcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. In this study, we have developed a modified YSD method that utilises SpyTag/Sp圜atcher-based in vivo protein ligation. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies.