Yeast surface display has been used as an effective tool to isolate and engineer traditional antibody fragments (scFv, Fab and single domain antibody). Pichia pastoris is the most commonly used yeast species in protein production. Here, Creative BioMart has developed a protocol for yeast surface display system developed in Pichia pastoris. In this system, the immune or mature library of single-domain antibody is fused with the C-terminal domain of Saccharomyces cerevisiae α-agglutinin gene (SAG1) and expressed on the cell surface of Pichia pastoris. Ligand labeling can be used for rapid quantitative analysis and separation of single-domain antibodies with desired characteristics.
To construct the cloning vector, the corresponding sequences of the single-domain antibody library need to be N-terminally fused to the pre-pro-sequence of the Saccharomyces cerevisiae α mating factor and C-terminally fused to the sequence encoding the last 320 amino acids (containing the GPI anchor attachment site) of the Saccharomyces cerevisiae α-agglutinin.
Please note that in addition to the vector we offered, clients can also provide their own or authorize us to purchase other expression vectors.
Recombinant plasmid will be linearized after sequence verification. Then the positive clones will be screened by electroporation. A library size of over 1 × 107 individual clones will be obtained.
Displayed antibodies format, antigen-binding activity will be conducted. The obtained library will be screened by high-speed flow cytometric sorting. Antigen binding clones will be isolated by classifying double staining cells on FACS. The clones with high affinity will be obtained after successive rounds of sorting by adjusting the sorting gate and lower concentration of ligand.
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