Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides.
Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides.
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DOI：
10.1016/j.jbiotec.2014.11.007
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发表时间：
2015-01
影响因子：
4.1
通讯作者：
Man Xu;Siyi Hu;Bo Ding;Caiyi Fei;Wen Wan;Dongmei Hu;Ruikai Du;Xiaochuan Zhou;Jiong Hong-Jiong-H
中科院分区：
文献类型：
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作者：
Man Xu;Siyi Hu;Bo Ding;Caiyi Fei;Wen Wan;Dongmei Hu;Ruikai Du;Xiaochuan Zhou;Jiong Hong-Jiong-H
我们报告了一个合理的策略，设计和构建多个小扰动诱变（SPM）库使用大规模并行合成的寡核苷酸的微芯片上的亲和力成熟的工程抗ErbB2抗体chA21。在全面分析Kabatman数据库中6个互补决定区（CDR）的序列和结构关系的基础上，开发了一种计算算法，使用模糊核苷酸将单位点和双位点突变引入可变CDR位置。6个SPM文库由419个简并寡核苷酸组成，其可以扩展成161，832个独特的CDR序列，具有95%天然氨基酸多样性的高覆盖率。我们使用Illumina下一代测序来证明合成CDR文库序列以及每个序列的相对量可以通过调整反应室分配和输入核苷组成来精确控制。利用微芯片合成的寡核苷酸，通过一步诱变PCR方法，构建了单链抗体噬菌体文库。大肠杆菌转化剂。具有来自四个单独CDR的组合突变的变体实现了超过19倍的亲和力增加。本文所述的策略应广泛适用于抗体和其他蛋白质的亲和力和选择性研究。
We report a rational strategy to design and construct multiple small perturbation mutagenesis (SPM) libraries using massively parallel synthesis of oligonucleotides on a microchip for affinity maturation of an engineered anti-ErbB2 antibody chA21. On the basis of a comprehensive analysis of the sequence and structural relationships of six complementary determination regions (CDRs) in the Kabatman database, a computational algorithm was developed to introduce single-site and double-site mutations into variable CDR positions using ambiguous nucleotides. The six SPM libraries were composed of 419 degenerate oligonucleotides that can be expanded into 161,832 unique CDR sequences with a high coverage ratio of 95% natural amino acid diversity. We used Illumina next-generation sequencing to demonstrate that the synthetic CDR library sequences, as well as relative quantities per sequence, can be controlled precisely by adjusting reaction chamber assignment and input nucleoside composition. The microchip-synthesized oligonucleotides were used for construction of single-chain antibody fragment (scFv) phage libraries through one-step mutagenic PCR of double-stranded plasmids with >106E. colitransformants. A variant with combinatorial mutations from four individual CDRs achieved more than 19-fold affinity increase. The strategy described herein should be broadly applicable to affinity and selectivity studies of antibodies and other proteins.