Human αB-crystallin (HSPB5) is frequently modified post-translationally by UV radiation, oxidation, and age-associated processes, which complicates functional analyses of the protein using natural sources. Thus, determining the biological function of HSPB5 at the molecular structure level requires unmodified protein. Here, we employed an Escherichia coli cell-free protein synthesis system to prepare unmodified, functionally active human HSPB5. An S30 extract prepared from E. coli strain BL21 (DE3) was used for HSPB5 synthesis. The efficacy of protein synthesis was assessed by monitoring influencing factors, such as the concentrations of Mg²⁺ and other reaction mixture constituents, and by evaluating batch and/or dialysis synthesis systems. Chaperone-like activity of synthesized HSPB5 was assayed using alcohol dehydrogenase (ADH) under thermal stress. The amount of HSPB5 synthesized using the cell-free system depended significantly on the concentration of Mg²⁺ in the reaction mixture. Use of condensed S30 extract and increased levels of amino acids promoted HSPB5 production. Compared with the batch system, HSPB5 synthesis was markedly increased using the dialysis system. The construction vector played a critical role in regulating the efficacy of protein synthesis. HSPB5 synthesized using the cell-free system had a native molecular mass, as determined by mass spectrometry analysis. The co-presence of synthesized HSPB5 suppressed heat-associated denaturation of ADH. Human HSPB5 synthesized using the cell-free system thus retains functional activity as a molecular chaperone.

中文翻译：

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无细胞合成功能活跃的HSPB5。

人类αB-晶状蛋白（HSPB5）经常通过紫外线辐射，氧化和与年龄相关的过程进行翻译后修饰，这使使用天然来源的蛋白质功能分析变得复杂。因此，在分子结构水平确定HSPB5的生物学功能需要未修饰的蛋白质。在这里，我们采用了大肠杆菌的无细胞蛋白质合成系统来制备未修饰的，具有功能活性的人HSPB5。从大肠杆菌菌株BL21（DE3）制备的S30提取物用于HSPB5合成。通过监测影响因素（例如Mg ²⁺的浓度）评估蛋白质合成的功效和其他反应混合物成分，并通过评估间歇和/或透析合成系统。使用乙醇脱氢酶（ADH）在热胁迫下测定合成的HSPB5的伴侣蛋白活性。使用无细胞系统合成的HSPB5的量很大程度上取决于Mg ²⁺的浓度在反应混合物中。浓缩S30提取物的使用和氨基酸水平的提高促进了HSPB5的产生。与批处理系统相比，使用透析系统可显着增加HSPB5的合成。构建载体在调节蛋白质合成的功效中起关键作用。使用无细胞系统合成的HSPB5具有天然分子量，这是通过质谱分析确定的。合成的HSPB5的共存抑制了ADH的热相关变性。因此，使用无细胞系统合成的人HSPB5保留了作为分子伴侣的功能活性。