Protein self-assemblies at the micro- and nano-scale are of great interest because of their morphological diversity and good biocompatibility. High-throughput screening of protein self-assembly at different scales and morphologies using protein crystallization screening conditions is an emerging method. When using this method to screen protein self-assembly conditions, some apparently transparent droplets are often observed, in which it is not clear whether self-assembly occurs. We explored the interaction between β-lactoglobulin and the protein crystallization kit Index™ C10 and observed the presence of micro- and nano-scale protein self-assemblies in the transparent droplets. The diverse morphology of the micro- and nano-scale self-assemblies in the transparent droplets formed by mixing different initial concentrations of β-lactoglobulin and Index™ C10 was further investigated by scanning electron microscope. Self-assembly process of fluorescence-labelled β-lactoglobulin was monitored continuously by laser confocal microscope, allowing real-time observation of the liquid-liquid phase separation phenomenon and the morphology of the final self-assemblies. The internal structure of the self-assemblies was gradually ordered over time by in-situ X-ray diffraction. This indicates that the self-assembly phenomenon within transparent droplets, observed in protein self-assembly condition screening experiments, is worthy of further in-depth exploration.
微-纳尺度的蛋白质自组装体具有形貌多样性与良好的生物相容性，因而成为蛋白质自组装领域的研究热点。以蛋白质结晶条件的筛选手段高通量筛选不同类型蛋白质于不同尺度、不同形貌的自组装过程，是一种新兴的研究方法，具有重要研究意义。利用该方法进行蛋白质自组装条件筛选时，常会形成一些表观透明的液滴，其中是否有自组装现象的发生尚不明确。文中以β-乳球蛋白与蛋白质结晶试剂盒Index™ C10相互作用为例进行探索，实验结果表明透明液滴中存在微-纳尺度的蛋白质自组装体。进一步通过扫描电镜观察不同初始浓度β-乳球蛋白与Index™ C10混合形成的透明液滴中微-纳自组装体的形貌有所差别；通过激光共聚焦显微镜连续拍摄添加荧光标签的β-乳球蛋白形成自组装体的过程，可实时观察到液液相分离现象及最终形成的自组装体的形貌；通过原位X-射线衍射手段，可观察到自组装体内部结构随时间推移逐渐有序化的过程。以上研究表明，在以结晶条件筛选手段为基础的蛋白质自组装条件筛选实验中，透明液滴内的自组装现象具有深入探索的必要和价值。.
Keywords: Index™ C10; clear droplets; micro- and nano-scale protein assembly; β-lactoglobulin.