Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.
Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.
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DOI：
10.1016/j.pep.2015.01.013
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发表时间：
2015-09
影响因子：
1.6
通讯作者：
Kontopidis, George
中科院分区：
文献类型：
--
作者：
Grigoroudis, Asterios I.;McInnes, Campbell;Premnath, Padmavathy Nandha;Kontopidis, George
关键词：
人类蛋白质的细菌表达仍然是蛋白质晶体学和药物设计中的关键挑战。虽然人细胞周期蛋白A构建体已被广泛表征为与细胞周期蛋白依赖性激酶2（CDK 2）的复合物，但在大肠杆菌中以稳定形式表达不含激酶亚基的单体人细胞周期蛋白A2的努力充满了技术困难，包括溶解性、产率和纯度。在这里，优化的条件进行了描述，目的是第一次产生，足够数量的人重组细胞周期蛋白A2的可溶性和活性形式的结晶和配体表征的目的。这些研究涉及在自诱导条件下并由分子伴侣表达质粒介导的His标记的异源表达系统的实施。通过与来自大肠杆菌的分子伴侣共表达，以天然折叠和可溶性形式获得了高产量的人细胞周期蛋白A2。大肠杆菌的各种组合。利用一步亲和层析法将融合蛋白产物纯化至均一，并通过对抑制肽的配体结合亲和力确认生物活性，所述抑制肽代表细胞周期蛋白调节亚基上的CDK 2底物募集位点的关键决定簇的替代物。总的来说，以直接和容易的方式获得没有CDK配偶体（在本工作中称为单体）的活性细胞周期蛋白A将解决CDK 2/细胞周期蛋白A复合物的蛋白质产生问题，并使得能够通过细胞周期蛋白沟进行非ATP竞争性CDK抑制的药物发现努力。
Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred as monomeric in this work) in a straightforward and facile manner will obviate protein –production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.
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基于伴侣的程序，以增加大肠杆菌中产生的可溶性重组蛋白的产量。
影响因子：
3.5
作者：
通讯作者：
--
影响因子：
4
作者：
Baumli S;Hole AJ;Noble ME;Endicott JA
通讯作者：
Endicott JA
影响因子：
7.3
作者：
Liu, Shu;Premnath, Padmavathy Nandha;McInnes, Campbell
通讯作者：
McInnes, Campbell
影响因子：
1.6
作者：
Ma, Na;Wei, Liujing;Hua, Qiang
通讯作者：
Hua, Qiang
DOI：
10.1073/pnas.96.8.4325
发表时间：
1999-04-13
影响因子：
11.1
作者：
Chen, YNP;Sharma, SK;Kaelin, WG
通讯作者：
Kaelin, WG