Promoting crystallization of antibody-antigen complexes via microseed matrix screening.
Promoting crystallization of antibody-antigen complexes via microseed matrix screening.
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DOI：
10.1107/s0907444910026041
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发表时间：
2010-08
期刊：
影响因子：
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通讯作者：
Gilliland GL
中科院分区：
文献类型：
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作者：
Obmolova G;Malia TJ;Teplyakov A;Sweet R;Gilliland GL
描述了微种子基质筛选在与IL-13复合的相关抗体的结晶中的应用。自引晶和交叉引晶都有助于促进成核和提高命中率。描述了微种子基质筛选在抗体-抗原复合物结晶中的应用，用于一组抗体，包括小鼠抗IL-13抗体C836、其人源化版本H2 L 6和H2 L 6的亲和力成熟变体M1295。这些抗体的Fab片段与抗原人IL-13复合结晶。三种复合物中的每一种的初始结晶筛选包括192个条件。对于H2 L 6仅观察到一次命中，而对于其他两种复合物未观察到命中。使用这些微晶的基质自微接种在各种条件下产生多次命中，所述条件被进一步优化以生长衍射质量的H2 L 6晶体。同样的H2 L 6晶种也成功地用于促进其他两种复合物的结晶。M1295晶体似乎与H2 L 6的晶体同晶，而C836晶体呈不同的晶体形式。这些结果与晶体生长的最佳条件可能不同于有利于成核的条件的概念一致。使用自接种或交叉接种方法的微种子基质筛选被证明是一种快速、稳健和可靠的方法，不仅用于结晶条件的细化，而且还促进晶体成核和增加命中率。
The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. The application of microseed matrix screening to the crystallization of antibody–antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate.