In vitro Evolution of Antibody Affinity via Insertional Mutagenesis Scanning of an Entire Antibody Variable Region
In vitro Evolution of Antibody Affinity via Insertional Mutagenesis Scanning of an Entire Antibody Variable Region
复制标题
通过整个抗体可变区的插入诱变扫描抗体亲和力的体外进化
DOI：
--
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
F. Hollfelder
中科院分区：
文献类型：
--
作者：
Kalliopi Skamaki;S. Emond;Matthieu Chodorge;J. Andrews;D. G. Rees;Daniel A. Cannon;B. Popovic;A. Buchanan;R. Minter;F. Hollfelder
我们报告的第一个系统的组合探索抗体的亲和力增强插入和缺失（InDels）。使用通过TRIAD方法的基于转座子的InDel的引入来产生具有跨越整个scFv基因的随机框内InDel的大文库，所述大文库进一步重组并通过核糖体展示筛选。从TRIAD文库中获得的潜在插入点的知识形成了通过插入扫描诱变（ISM）探索新插入的长度和序列多样性的基础。作为InDel诱变和与已知点突变组合的结果，抗IL-13抗体BAK 1的总体256倍亲和力改善验证了这种方法，并表明这种InDel方法和点突变的常规探索的结果可以协同作用以产生具有更高亲和力的抗体。插入/缺失（InDel）突变在基因组和蛋白质进化中起着关键作用。尽管它们在进化史上的突出地位，InDels通过定向进化改变蛋白质工程功能的潜力仍然未被探索。相反，点诱变被广泛使用。在这里，我们创建了含有InDel的抗体文库，并证明可以以这种方式实现亲和力成熟，建立了所有先前体外选择中采用的点突变策略的替代方案。这些InDel反映了在源自相同种系基因的天然抗体的环中观察到的相当大的长度变化，并与点突变相结合，使得功能创新的天然来源可用于试管中的人工进化。
We report the first systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD was used to generate large libraries with random in-frame InDels across the entire scFv gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (ISM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach and suggests that the results of this InDel approach and conventional exploration of point mutations can synergize to generate antibodies with higher affinity. Significance Insertion/deletion (InDel) mutations play key roles in genome and protein evolution. Despite their prominence in evolutionary history, the potential of InDels for changing function in protein engineering by directed evolution remains unexplored. Instead point mutagenesis is widely used. Here we create antibody libraries containing InDels and demonstrate that affinity maturation can be achieved in this way, establishing an alternative to the point mutation strategies employed in all previous in vitro selections. These InDels mirror the observation of considerable length variation in loops of natural antibodies originating from the same germline genes and be combined with point mutations, making both natural sources of functional innovation available for artificial evolution in the test tube.
登录
查看更多内容
影响因子：
5.6
作者：
YANG, WP;GREEN, K;BARBAS, CF
通讯作者：
BARBAS, CF
通过插入/删除诱变访问定向酶进化中序列空间中未探索的区域
DOI：
10.17863/cam.72969
发表时间：
2020
期刊：
--
影响因子：
--
作者：
Emond S
通讯作者：
Emond S
影响因子：
30.3
作者：
Kepler TB;Liao HX;Alam SM;Bhaskarabhatla R;Zhang R;Yandava C;Stewart S;Anasti K;Kelsoe G;Parks R;Lloyd KE;Stolarchuk C;Pritchett J;Solomon E;Friberg E;Morris L;Karim SS;Cohen MS;Walter E;Moody MA;Wu X;Altae-Tran HR;Georgiev IS;Kwong PD;Boyd SD;Fire AZ;Mascola JR;Haynes BF
通讯作者：
Haynes BF
DOI：
10.1073/pnas.91.9.3809
发表时间：
1994-04-26
影响因子：
11.1
作者：
BARBAS, CF;HU, D;BURTON, DR
通讯作者：
BURTON, DR
影响因子：
8.8
作者：
Scharf L;Scheid JF;Lee JH;West AP Jr;Chen C;Gao H;Gnanapragasam PN;Mares R;Seaman MS;Ward AB;Nussenzweig MC;Bjorkman PJ
通讯作者：
Bjorkman PJ