Developing Antibodies Using Yeast

Yeast surface display or yeast display is a powerful tool used for isolating and engineering antibodies, increasing their specificity, affinity, and stability. It has been used to engineer antibodies to target various antigens including; huntingtin protein, T cell receptors, carcinoembryonic antigen, and botulinum neurotoxin.

The antibody isolation and engineering process

The single-chain fragment variable (scFv) is the smallest human antibody fragment with binding function. In the yeast surface display, the antibody is displayed in scFv form, in which the variable regions of the heavy (VH) and light chains (VL) of the antibody are connected together with a short flexible linker polypeptide.

The yeast (Saccharomyces cerevisiae) cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p).

Through disulfide bonds to Aga1p attaching the yeast cell wall, the scFv is joined to the adhesion subunit (Aga2p) of the yeast agglutinin protein.

The expression of the Aga2pscFv is under the control of a galactose-inducible promoter on the yeast display plasmid. Each yeast cell normally displays 1×104 to 1×105 copies of the scFv.

Using an existing nonimmune human library, the scFv is isolated with the help of magnetic-activated cell sorting and selection via flow cytometry.

Then, this enriched population is mutagenized, and consecutive rounds of random mutagenesis and flow cytometry selection are performed to reach the required scFv characters via directed evolution.

Advantages of yeast surface display

Yeast surface display has various benefits for protein directed evolution. For example, quantitative screening is enabled in yeast surface display via fluorescence-activated cell sorting (FACS), allowing the equilibrium activity and sample statistics to be monitored directly during the screening process.

The antigen-binding signal is also normalized for expression, removing artifacts due to host expression bias, allowing for fine discrimination between mutants.

Engineered antibodies, using surface display yeast, have high stability as the expression is calculated directly and correlated with the stability of the displayed protein.

After maturation, the affinity of an antibody can be appropriately ‘titrated’ while displayed on the yeast surface, avoiding the necessity for expression and purification of each clone. Boder and colleagues used yeast surface display to engineer an antibody to fluorescein with femtomolar affinity, the highest affinity reported so far.

The binding properties on the yeast surface are similar to those measured in solution or by biosensor techniques. Endoplasmic reticulum chaperones and quality-control ‘machinery’ allow the displayed proteins to be folded in the endoplasmic reticulum of the eukaryotic yeast cells. No other display libraries are able to provide complex post-translational modifications to proteins.

Disadvantages of yeast surface display

The potentially smaller functional library size than that of other selection techniques is a theoretical limitation of yeast surface display.

Yet, it is hard to accurately calculate the real functional diversity of any display library, and bias-free propagation of yeast libraries has been found over-amplification of 1010-fold. Moreover, all other techniques usually under-sample the theoretical sequence space of scFv.

In yeast surface display, the conformation of VH and VL domains in scFv may not be similar to its natural IgG, in which the heavy and light chains also bind through their constant heavy 1 (CH1) and constant light (CL) domains.

Although these conformational variations are usually small, their effect on binding affinity can be serious and troublesome for affinity maturation studies. It is common that loss of significant potency happens when an affinity matured scFv clone is converted back to its associated IgG.

Recent trends in yeast surface display

Since the Fab domain (antigen-binding fragment) involves an entire light chain (VL-CL) and half heavy chain (VH-CH1), this format can keep VH and VL domains in their whole conformations.

Thus, it is more plausible to say the ideal combination for affinity maturation is to display Fabs on yeast surface and to screen by FACS.

Recent researches reported the feasibility of displaying Fabs and full-length IgGs on the yeast cell surface. Researchers concluded that Fab was more reliable than scFv and Fab was appropriate for antibody affinity maturation.

Sources

  • Boder, E. T., & Wittrup, K. D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology, 15(6), 553-557.
  • Boder, E. T., Midelfort, K. S., & Wittrup, K. D. (2000). Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proceedings of the National Academy of Sciences, 97(20), 10701-10705.
  • Chao, G., Lau, W. L., Hackel, B. J., Sazinsky, S. L., Lippow, S. M., & Wittrup, K. D. (2006). Isolating and engineering human antibodies using yeast surface display. Nature protocols, 1(2), 755.
  • Gai, S. A., & Wittrup, K. D. (2007). Yeast surface display for protein engineering and characterization. Current opinion in structural biology, 17(4), 467-473.
  • Mei, M., Li, J., Wang, S., Lee, K. B., Iverson, B. L., Zhang, G., ... & Yi, L. (2019). Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression. Front. Bioeng. Biotechnol. 7: 362. DOI: 10.3389/fbioe.
  • Shusta, E. V., Pepper, L. R., Cho, Y. K., & Boder, E. T. (2008). A decade of yeast surface display technology: where are we now?. Combinatorial chemistry & high throughput screening, 11(2), 127-134.
  • Sivelle, C., Sierocki, R., Ferreira-Pinto, K., Simon, S., Maillere, B., & Nozach, H. (2018, July). Fab is the most efficient format to express functional antibodies by yeast surface display. In MAbs (Vol. 10, No. 5, pp. 720-729). Taylor & Francis.

Further Reading

Last Updated: Jan 28, 2021

Dr. Ahmed Donia

Written by

Dr. Ahmed Donia

Since his early childhood, Ahmed has such an inquisitive mind that cannot stop searching for answers. His mind has always been skeptic and questionable. He has never been satisfied with simple answers such as “Yes” or “No”. Ahmed has a BSc in Pharmaceutical Sciences and an MSc in Microbiology. Currently, he is a Ph.D. candidate in Microbiology and Immunology.

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