Ligand supplementation as a method to increase soluble heterologous protein production
Ligand supplementation as a method to increase soluble heterologous protein production
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配体补充作为增加可溶性异源蛋白产量的方法
DOI：
10.1586/14789450.5.1.137
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发表时间：
2008
影响因子：
3.4
通讯作者：
U. Oppermann
中科院分区：
文献类型：
--
作者：
V. Hozjan;K. Guo;Xiaoqiu Wu;U. Oppermann
配体相互作用是酶或受体功能的核心，构成了生物化学和药理学的基石。在这里，我们讨论了配体的应用，可以利用显着增加的比例重组蛋白表达的可溶性形式，包括在培养过程中的配体。只要有足够可溶的、细胞可渗透的和亲合力的配体，就可以用它来稳定初始合成的蛋白质，并以这种方式促进溶解度和防止聚集。据我们所知，这一概念尚未得到系统的探讨，我们在这里提供的第一个数据配体补充在整个人类蛋白质家族的表达实验：短链淀粉酶/还原酶（SDR）。我们确定了甘油rhitinic酸和它的半琥珀酸酯，甘珀酸（CBX），作为配体的可变亲和力范围从低纳摩尔到微摩尔的结合常数对几个SDR。CBX被用作大肠杆菌表达系统中的培养添加剂，针对来自65个SDR靶标的总共约500个构建体，并且对于超过四个不同的靶标获得了显著更高水平的可溶性蛋白。其中之一，糖皮质激素激活酶1型11 β-羟基类固醇脱氢酶（11 β-HSD1），在不存在配体的情况下仅以非常低的水平（<10 µ g/l培养物）可溶性表达;然而，通过包含CBX或其他抑制剂，可溶性表达可增强至mg/l水平。在等效表达实验中使用具有不同化学支架的其他化合物对抗11 β-HSD1，得到类似结果。总之，如果给定蛋白质的合适配体是可用的，这种方法可以快速测试，并可能代表一种简单有效的策略，以提高可溶性蛋白质的生产，适合结构和功能表征研究。
Ligand interactions are central to enzyme or receptor function, constituting a cornerstone in biochemistry and pharmacology. Here we discuss a ligand application that can be exploited to significantly increase the proportion of recombinant protein expressed in soluble form, by including ligands during the culture. Provided that a sufficiently soluble, cell-permeable and avid ligand is available, one can use it to stabilize nascently synthesized proteins, and in this manner promote solubility and prevent aggregation. To our knowledge, this concept has not been explored systematically and we provide here the first data on ligand supplementation in expression experiments across a whole human protein family: the short-chain dehydrogenases/reductases (SDR). We identified glycerrhitinic acid and its hemisuccinate ester, carbenoxolone (CBX), as ligands with variable affinities ranging from low nanomolar to micromolar binding constants against several SDRs. CBX was utilized as a culture additive in Escherichia coli expression systems against a total of approximately 500 constructs derived from 65 SDR targets, and significantly higher levels of soluble protein were obtained for more than four distinct targets. One of these, the glucocorticoid-activating enzyme type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1), was solubly expressed only at a very low level (<10 µg/l culture) in the absence of ligand; however, soluble expression could be enhanced to mg/l levels by inclusion of CBX or other inhibitors. Other compounds with different chemical scaffolds were used against 11β-HSD1 in equivalent expression experiments yielding similar results. Taken together, if suitable ligands for a given protein are available, this approach could be tested quickly and might represent an easy and effective strategy to enhance soluble protein production, suitable for structural and functional characterization studies.