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Isolating and engineering human antibodies using yeast surface display

Nature Protocols volume 1, pages 755–768 (2006)Cite this article

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Abstract

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.

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Figure 1: Yeast surface display.
Figure 2
Figure 3: Representative flow cytometry data.
Figure 4: Overlay of representative flow cytometry data for two scFv clones.
Figure 5: Titration curves.

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Change history

  • 06 September 2007

    In the version of this article initially published, the x-axis of Figure 4b should have been labeled “PE antigen binding”, not “Alexa Fluor 488 (c-Myc)”. The figure has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by CA96504, CA101830 and AI065824 from the National Institutes of Health, a David Koch Graduate Fellowship from the Massachusetts Institute of Technology Center for Cancer Research (G.C.), National Defense Science and Engineering Graduate Fellowships (G.C. and B.J.H.), a National Institute of General Medical Sciences Biotechnology Training Grant (S.L.S.) and a National Science Foundation Graduate Fellowship (S.M.L.). We thank the Massachusetts Institute of Technology Flow Cytometry Core Facility for their assistance, and E. Boder, J. Cochran, M. Feldhaus, M. Roguska and E. Shusta for detailed feedback on this protocol manuscript.

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Authors and Affiliations

  1. Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue E19-563, Cambridge, 02139, Massachusetts, USA

    Ginger Chao, Wai L Lau, Benjamin J Hackel, Stephen L Sazinsky, Shaun M Lippow & K Dane Wittrup

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  1. Ginger Chao
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  2. Wai L Lau
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  3. Benjamin J Hackel
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  4. Stephen L Sazinsky
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  6. K Dane Wittrup
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Contributions

G.C., Introduction, labeling and enrichment protocols, Anticipated Results, Figs. 1, 2 and 4, and assembly of manuscript; W.L.L., Box 1, Fig. 3 and Supplementary Fig. 1; B.J.H., characterization, mutagenesis and transformation protocols and Fig. 3; S.L.S., MACS protocol; S.M.L., labeling and titration protocols, Figs. 4 and 5 and Table 1; and all authors participated in discussions on optimal protocol parameters and the editing and revision of the manuscript.

Corresponding author

Correspondence to K Dane Wittrup.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

pCTCON2 plasmid map. (PDF 92 kb)

Supplementary Table 1

Preferred yeast culturing vessels. (PDF 104 kb)

Supplementary Methods (PDF 39 kb)

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Chao, G., Lau, W., Hackel, B. et al. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1, 755–768 (2006). https://doi.org/10.1038/nprot.2006.94

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