用奇米蛋白构建功能蛋白区的鉴定
Summary
结构上相关的蛋白质经常发挥不同的生物学功能。交换这些蛋白质的等效区域, 以创造嵌合蛋白是一种创新的方法, 以确定关键的蛋白质区域, 负责其功能分化。
Abstract
该协议的目标包括设计嵌合蛋白, 在这种蛋白质的不同区域被结构相似的蛋白质中的相应序列所取代, 以确定这些区域的功能重要性。这种嵌合体是通过嵌套 pcr 协议产生的, 该协议使用重叠的 dna 片段和经过充分设计的引物, 然后在哺乳动物系统中表达, 以确保本地二级结构和翻译后的修饰。
然后, 在适当的读出分析中, 嵌合体的活性丧失表明了不同区域的功能作用。因此, 确定了隐藏一组关键氨基酸的区域, 这些区域可以通过互补技术 (例如位向诱变) 进一步筛选, 以提高分子分辨率。虽然仅限于可以发现功能不同的结构相关蛋白质的情况, 但嵌合蛋白已被成功地用于识别蛋白质 (如细胞因子和细胞因子受体) 中的关键结合区域。这种方法特别适用于蛋白质的功能区域没有得到很好界定的情况, 是定向进化方法的宝贵的第一步, 目的是缩小感兴趣的区域, 减少所涉及的筛选工作。
Introduction
几种类型的蛋白质, 包括细胞因子和生长因子, 被分组在家庭的成员有相似的三维结构, 但往往发挥不同的生物功能1,2。这种功能多样性通常是分子活性位点3中氨基酸组成差异小的结果.确定这些位点和功能决定因素不仅提供了宝贵的进化见解, 而且还设计了更具体的激动剂和抑制剂4。然而, 在与结构相关的蛋白质之间经常发现的残留物成分的大量差异使这项任务变得复杂。尽管构建包含数百个突变体的大型库现在是可行的, 但评估每一个残留物的变化和组合仍然是一项具有挑战性和耗时的工作5。
因此, 评估大型蛋白质区域的功能重要性的技术对于将可能的残留物数量减少到可管理的第6种是有价值的.截断蛋白一直是解决这一问题最常用的方法。因此, 如果所研究的蛋白质功能受到特定区域7、8、9的删除的影响, 则区域在功能上被认为是相关的。然而, 这种方法的一个主要局限性是, 缺失会影响蛋白质的二级结构, 导致错误折叠、聚集和无法研究预期的区域。一个很好的例子是细胞因子在成本蛋白 m (osm) 上的截断版本, 其中一个内部删除大于7个残留物导致一个错误折叠的突变体, 不能进一步研究10。
嵌合蛋白的产生是一种替代和创新的方法, 允许分析更大的蛋白质区域。这种方法的目的是通过另一种蛋白质中与结构相关的序列交换对蛋白质感兴趣的区域, 以评估被替换的部分对特定生物功能的贡献。在信号受体领域广泛用于识别功能域11,12, 嵌合蛋白是特别有用的研究蛋白质家族的氨基酸认同少, 但保守的二级结构。在白细胞介素-6 (il-6) 类细胞因子类中可以找到适当的例子, 如白细胞介素-6 和睫毛神经营养因子 (6% 序列识别)13或白血病抑制因子 (lif) 和 osm (20% 身份)6, 其中下面的协议是基于。
Protocol
1. 奇美蛋白设计 选择合适的蛋白质 (供体) 与感兴趣的蛋白质 (接受者) 交换区域捐献者蛋白应该在结构上相似, 理想的情况下属于同一个蛋白质家族, 但缺乏生物活性作为读出。如果不知道与结构相关的蛋白质, 则可以使用自动工具 (如矢量对齐搜索工具 (vast)14、15)来识别潜在的候选元素 访问蛋白质数据库 (pdb)16欧洲网站 (h…
Representative Results
嵌合蛋白的构建和生成 (图 1) 将以白细胞介素-6 细胞因子家族的两名成员 osm 和 lif 为例,这是最近发表的一项研究的主题6。图 2显示了这些蛋白质的三维结构。这两个分子都采用 i 类细胞因子的特征二级结构, 四个螺旋 (称为 a 到 d) 被捆绑起来, 并由循环 28连接。人类蛋白质的对齐氨基酸结构?…
Discussion
嵌合蛋白的产生构成了一种多功能的技术, 它能够超越截断蛋白的限制, 以解决细胞因子受体结合域的模块化等问题13。嵌合体的设计是这种研究的关键一步, 需要仔细考虑。建立功能域的初步研究一般需要在第一阶段替换广泛的区域, 而较小的可变长度的替换则更适合于对单一区域的详细研究。在这一步中, 应特别注意蛋白质家族中存在的小的保守图案, 因为这些图案往往表明功?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了马克斯·普朗克协会和舒赫特曼诊所 (德国巴特罗滕费尔德) 的支持。这项研究的一部分最初发表在《生物化学》杂志上。aderan-segarra, j. m., schindler, n., gajawada, p., lörchner, h., braun, t. & pöling, j。人组织受体激活需要人 oncostatin m (osm) 结合部位 iii 中的 ab 环路和 d-螺旋。j. biol. chem. 2018;18: 7017-7029. ©作者。
Materials
| Labcycler thermocycler | Sensoquest | 011-103 | Any conventional PCR machine can be employed to carry out this protocol |
| NanoDrop 2000c UV-Vis spectrophotometer | ThermoFisher Scientific | ND-2000C | DNA quantification |
| GeneRuler 100 bp DNA ladder | ThermoFisher Scientific | SM0241 | |
| GeneRuler DNA Ladder Mix | ThermoFisher Scientific | SM0331 | |
| AscI restriction enzyme | New England Biolabs | R0558 | |
| PacI restriction enzyme | New England Biolabs | R0547 | |
| Phusion Hot Start II DNA Polymerase | ThermoFisher Scientific | F-549S | |
| dNTP set (100 mM) | Invitrogen | 10297018 | |
| T4 DNA ligase | Promega | M1804 | |
| NucleoSpin Gel and PCR clean-up kit | Macherey-Nagel | 740609 | |
| MGC Human LIF Sequence-Verified cDNA (CloneId:7939578), glycerol stock | ThermoFisher Scientific | MHS6278-202857165 | |
| LE agarose | Biozym | 840004 | |
| Primers | Sigma-Aldrich | Custom order | |
| Human Oncostatin M cDNA | Gift of Dr. Heike Hermanns (Division of Hepatology, University Hospital Würzburg, Germany) | ||
| pCAGGS vector with PacI and AscI restriction sites | Gift of Dr. André Schneider (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany) |
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Citar este artículo
Adrian-Segarra, J. M., Lörchner, H., Braun, T., Pöling, J. Identification of Functional Protein Regions Through Chimeric Protein Construction. J. Vis. Exp. (143), e58786, doi:10.3791/58786 (2019).