Abstract
Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) by converting deoxycytidines (dC) to deoxyuracils (dU) which then can induce other mutations, and plays a central role in introducing diversification of the antibody repertoire in B cells. Ectopic expression of AID in bacteria and non-B cells can also lead to frequent mutations in highly expressed genes. Taking advantage of this feature of AID, in recent years, systems coupling in vitro somatic hypermutation and mammalian cell surface display have been developed, with unique benefits in antibody discovery and optimization in vitro. Here, we provide a protocol for AID mediated in vitro protein evolution. A CHO cell clone bearing a single gene expression cassette has been constructed. The gene of an interested protein for in vitro evolution can be easily inserted into the cassette by dual recombinase-mediated cassette exchange (RMCE) and constantly expressed at high levels. Here, we matured an anti-TNFα antibody as an example. Firstly, we obtained a CHO cell clone highly displaying the antibody by dual RMCE. Then, the plasmid expressing AID is transfected into the CHO cells. After a few rounds of cell sorting-cell proliferation, mutant antibodies with improved features can be generated. This protocol can be applied for improving protein features based on displaying levels on cell surface and protein-protein interaction, and thus is able to enhance affinity, specificity, and stability besides others.
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Acknowledgments
This work was supported by the grant from Ministry of Science and Technology of the People’s Republic of China (grant number 2014CB910402) and the National Natural Science Foundation of China (grant number 31370792).
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An, L., Chen, C., Luo, R., Zhao, Y., Hang, H. (2018). Activation-Induced Cytidine Deaminase Aided In Vitro Antibody Evolution. In: Liu, C. (eds) B Cell Receptor Signaling. Methods in Molecular Biology, vol 1707. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7474-0_1
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DOI: https://doi.org/10.1007/978-1-4939-7474-0_1
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7474-0
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