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. 2019 Feb 25;400(3):383-393.
doi: 10.1515/hsz-2018-0347.

Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning

Lukas Roth  1 , Julius Grzeschik  1 , Steffen C Hinz  1 , Stefan Becker  2 , Lars Toleikis  2 , Michael Busch  3 , Harald Kolmar  1 , Simon Krah  2 , Stefan Zielonka  2
Affiliations

Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning

Lukas Roth et al. Biol Chem. .

Abstract

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

Keywords: Golden Gate Cloning; antibody discovery; chimeric antibodies; gap repair cloning; human antibodies; yeast mating.

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References

    1. Abdiche, Y.N., Harriman, R., Deng, X., Yeung, Y.A., Miles, A., Morishige, W., Boustany, L., Zhu, L., Izquierdo, S.M., and Harriman, W. (2016). Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms. MAbs 8, 264–277.
    1. Barbas, C.F., Kang, A.S., Lerner, R.A., and Benkovic, S.J. (1991). Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982.
    1. Benatuil, L., Perez, J.M., Belk, J., and Hsieh, C.-M. (2010). An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng. Des. Sel. PEDS 23, 155–159.
    1. Boder, E.T. and Wittrup, K.D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557.
    1. Braunstein, G.D., Rasor, J., Danzer, H., Adler, D., and Wade, M.E. (1976). Serum human chorionic gonadotropin levels throughout normal pregnancy. Am. J. Obstet. Gynecol. 126, 678–681.

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