Advancing Pir-based yeast surface display by reshuffling and truncating Pir2-β-lactamase constructs (CROSBI ID 724529)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa
Podaci o odgovornosti
Žunar, Bojan ; Lozančić, Mateja ; Matičević, Ana ; Matijević, Dominik ; Martinić Cezar, Tea ; Teparić, Renata ; Mrša, Vladimir
engleski
Advancing Pir-based yeast surface display by reshuffling and truncating Pir2-β-lactamase constructs
After synthesizing proteins, yeast Saccharomyces cerevisiae can retain them within the cell, secrete them into the medium, or bind them to the cell surface. In the third case, yeast anchors proteins by covalently binding them to β-glucan residues, thus allowing for surface display, a cell-engineering technique that can remake the entire cell surface into catalytically active living material. To immobilize proteins of interest closer to their N-terminus, the proteins are regularly fused with one of five Pir proteins (proteins with internal repeats), which covalently bind to β-1, 3-glucan. However, beyond the requirement for glutamine in their characteristic internal repeats, the mechanism of attaching these proteins to the yeast cell wall remains unclear. Thus, the Pir-based surface display continues to rely on guesswork and intuition, which is less than efficient. To address this issue, we inserted β-lactamase at five positions in Pir2 (Hsp150) protein and followed its activity and Pir2-binding efficiency through enzymatic and immunochemical methods. Moreover, we constructed and tested additional six truncated Pir2-β-lactamase variants, thus determining the minimal portion of Pir2 required for efficient binding to the yeast cell wall. Finally, to enrich our experimental results with structural insights, we used a deep- learning Alphafold2-based algorithm to in silico predict the structure of Pir2-β-lactamase fusions. Therefore, we present and rationalize a novel set of practical guidelines for a reproducible, straightforward, and efficient Pir-based yeast surface display.
Pir proteins, cell wall, yeast, surface display, Saccharomyces cerevisiae
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
Podaci o prilogu
3-3.
2022.
objavljeno
Podaci o matičnoj publikaciji
British Yeast Group 2022: From Cells to Genomes: Poster Book
Podaci o skupu
British Yeast Group Meeting 2022: From Cells to Genomes
poster
07.09.2022-09.09.2022
London, Ujedinjeno Kraljevstvo