Department of Chemistry and Biochemistry, California State University Fresno, Fresno, CA, USA.
Methods Mol Biol. 2022;2446:513-530. doi: 10.1007/978-1-0716-2075-5_26.
VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure-function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex.
VHH 是从小型骆驼科动物抗体中克隆的抗原结合结构域。这些蛋白作为研究试剂、结晶辅助剂和治疗剂，在多种应用中引起了广泛关注。VHH 的进化适应性导致其具有独特的生物物理性质和抗原结合模式，与传统抗体不同。详细的 VHH 与同源蛋白抗原相互作用的分子分析对于理解结构-功能关系和蛋白质工程非常有价值。大多数 VHH 结合折叠蛋白，因此识别不连续的三维表位。虽然有多种方法可用于剖析蛋白抗原与 VHH 之间的相互作用，但 X 射线晶体学仍然是目前分辨率最高的方法。在这里，我们提供了一种确定和分析 VHH 与蛋白抗原复合物的 X 射线结构的更新程序。我们描述了 VHH 和蛋白抗原的重组表达和纯化、蛋白复合物的纯化和分析、结晶和优化、通过分子置换确定 X 射线结构以及复合物的分析。
