Optimization of the antibody CH3 domain by residue frequency analysis of IgG sequences
Optimization of the antibody CH3 domain by residue frequency analysis of IgG sequences
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DOI：
10.1016/j.jmb.2003.10.040
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发表时间：
2004-01-02
影响因子：
5.6
通讯作者：
Hansen, G
中科院分区：
文献类型：
--
作者：
Demarest, SJ;Rogers, J;Hansen, G
为了提高重组抗体蛋白的整体组装、产量和半衰期，我们克隆并表达了几个IgG1 C(H)3结构域，并研究了它们的折叠/重折叠特性。我们利用一个含有硫氧还蛋白还原酶敲除菌株BL21（DE3）的细胞质细菌表达系统来生产牛、鼠和人的C(H)3。在相同的条件下，表达牛C(H)3一致导致适当折叠/氧化蛋白的最高产量。圆形二色性和荧光实验表明，考虑到两个分子之间的边缘序列守恒，氧化牛和小鼠的C(H)3具有惊人的相似结构和稳定性。利用来自19种不同哺乳动物物种的36个独特Fc序列的有限数据集进行残基频率分析，针对牛C(H)3的5个特定位点进行优化。其中三种突变体的组合将分子的热稳定性提高到86度。将该方法与使用更大的序列数据库和/或不同选择标准的类似研究进行比较，表明序列数据库设计可以提高识别值得优化的残基位点的成功率。优化后的C(H)3结构域可以作为一个特别稳定的功能设计平台，并且可以嫁接到全长抗体序列中，以提高它们的热力学参数和保质期。(C) 2003 Elsevier Ltd.版权所有。
In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 C(H)3 domains and examined their folding/ refolding characteristics. We utilized a cytoplasmic bacterial expression system with a thioredoxin reductase knock-out strain of BL21(DE3) to produce bovine, murine and human C(H)3. Under identical conditions, expression of bovine C(H)3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C(H)3 have surprisingly similar structures and stabilities, considering the marginal sequence conservation between the two molecules. Residue frequency analysis using a limited data set of 36 unique Fc sequences originating from 19 different mammalian species targeted five specific sites for optimization within bovine C(H)3. Combination of three of these mutants increased the thermal stability of the molecule to 86 degreesC. Comparison of this approach to similar studies using larger sequence databases and/or different selection criteria suggests sequence database design can increase the success rate for identifying residue sites worth optimizing. This optimized C(H)3 domain can be used as a particularly stable platform for functional design and can be grafted into full-length antibody sequences to enhance their thermodynamic parameters and shelf-life. (C) 2003 Elsevier Ltd. All rights reserved.