| Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example
|
|
Author:
Date:
2017-09-05
[Abstract] The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. ...
[摘要] 与共激活因子p300的转录因子缺氧诱导因子（HIF）复合物，激活缺氧反应途径并允许肿瘤生长。每个相应蛋白质的CH1和CAD结构域形成p300和HIF之间的界面。正在开发靶向和抑制HIF / p300复合物形成的小分子化合物，目的是减少肿瘤生长。研究配体与p300-CH1相互作用的高分辨NMR光谱是必要的，为了进行结构和生物物理学研究，需要净化大量正确折叠的p300-CH1。 p300-CH1具有3个锌指和9个半胱氨酸残基，构成与试剂相容性和蛋白氧化相关的挑战。已经开发了一种通过在表达过程中并入锌并简化纯化时间来克服这些问题的方案，导致适合于结构NMR研究的最佳折叠蛋白质（120mg / 4L表达介质）的高产率。已证实最终重组p300-CH1的结构完整性是使用一维1 H NMR光谱和圆二色性最优的。该方案适用于纯化其他含锌指蛋白质。
【背景】由于不适当的血管灌注，实体瘤的发展与缺氧区的发展有关。对于缺氧微环境，肿瘤细胞过表达低氧诱导因子（HIF），一种异二聚体转录因子家族（Semenza，2002; Brat和Van Meir，2004; Kaur等，2005）。 HIFs结合p300（一种转录共激活因子），形成诱导HIF靶基因的复合物，从而激活缺氧反应途径并促进肿瘤生长（Kasper and Brindle，2006; Liu，2008）。涉及HIF / p300蛋白 ...
|
|
|
| Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)
|
|
Author:
Date:
2016-07-20
[Abstract] The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to ...
[摘要] 该协议描述了来自单纯疱疹病毒1和伪狂犬病病毒的核出口复合物（NEC）的可溶形式的产生和结晶。 NEC是由保守蛋白UL31和UL34组成的异源二聚体。 NEC低聚使受感染细胞中的衣壳周围的内核膜变形，从而在核出口期间介导衣壳发芽进入核周空间。 我们已经成功地开发了一个协议，从大规模制备高纯度NEC从两种不同的病毒在原核表达系统，这使我们能够结晶这些病毒蛋白复合物和确定其结构。 该程序可适于纯化和结晶其它可溶性蛋白质复合物。
|
|
|
| Construction of Glycine Oxidase Mutant Libraries by Random Mutagenesis, Site Directed Mutagenesis and DNA Shuffling
|
|
Author:
Date:
2014-10-05
[Abstract] Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate (Pedotti et al., 2009). Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of ...
[摘要] 草甘膦，广泛应用于全世界农业的广谱除草剂在莽草酸途径中抑制5-烯醇丙酮酸莽草酸-3-磷酸合酶，并且已经报道甘氨酸氧化酶（GO）能够催化各种胺的氧化脱氨基， 切割草甘膦中的CN键（Pedotti等人，2009）。 在这里，为了改善从草甘膦降解的蜡状芽孢杆菌菌株（BceGO）克隆的甘氨酸氧化酶的催化活性，我们使用基于噬菌体T7裂解的方法， 通过筛选氧化酶活性并通过定向进化改造编码BceGO的基因。
|
|
|