both the point mutations (K10E, T82I and E156G), and the deletion of mAID's NES contributed to the improvement of mAID activity (Fig. 2B). We also constructed hAID-del (human AID without NES) and hAID-plus (hAID-del with the point mutations K10E, T82Iand E156G) and tested their mutation efficiencies (Fig. 2B). Generally speaking, hAID and their mutants had lower activities than their mouse counterparts in CHO cells. The mutations K10E, T82I and E156G on hAID increased its activity, while in contrast to mAID, the NES deletion of hAID did not enhance its activity. These data suggest that mAID-plus has the highest mutating activity, and should be used for antibody affinity maturation in the following experiments.
Antibodies have been extensively used for the purpose of scientific research, clinical diagnosis, and therapy. Combination of in vitro somatic hypermutation and mammalian cell surface display has been an efficient technology for antibody or other proteins optimization, in which the efficiency of activation-induced cytidine deaminase (AID) mutations in genes is one of the most important key factors. Gene transcriptional level has been found to be positively proportional to AID-induced mutation frequency. Thus, construction of the cell clone bearing a gene of interest (GOI) with high transcription level can increase AID-induced mutations. In this study, a retargetable gene cassette is inserted onto predetermined chromosome site (ywhae gene site) which is among the genes with the highest as well as stable transcription, and is found that one subsite is suitable to be retargeted for efficient protein display in Chinese hamster ovary (CHO) cells. The resultant cell clone (T31) has higher and more stable transcription/expression than CHO-puro clone which was previously established through the strategy of random insertion followed by a high-throughput selection. It also possesses a significantly higher mutation frequency to GOI than CHO-puro cells; thus, it is a better clone for the in vitro improvement of antibody affinity, and probably other properties.
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