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An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression
Biochemistry ( IF 2.9 ) Pub Date : 2018-07-13 00:00:00 , DOI: 10.1021/acs.biochem.7b01081 Steven K Albanese 1, 2 , Daniel L Parton 2 , Mehtap Işık 2, 3 , Lucelenie Rodríguez-Laureano 2 , Sonya M Hanson 2 , Julie M Behr 2, 4 , Scott Gradia 5 , Chris Jeans 5 , Nicholas M Levinson 6 , Markus A Seeliger 7 , John D Chodera 2
Biochemistry ( IF 2.9 ) Pub Date : 2018-07-13 00:00:00 , DOI: 10.1021/acs.biochem.7b01081 Steven K Albanese 1, 2 , Daniel L Parton 2 , Mehtap Işık 2, 3 , Lucelenie Rodríguez-Laureano 2 , Sonya M Hanson 2 , Julie M Behr 2, 4 , Scott Gradia 5 , Chris Jeans 5 , Nicholas M Levinson 6 , Markus A Seeliger 7 , John D Chodera 2
Affiliation
Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 μg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene.
中文翻译:
用于自动化细菌表达的人激酶结构域构建的开放库
激酶在细胞信号传导中发挥着关键作用,并且在许多疾病中失调,例如癌症、糖尿病和神经退行性疾病。目前,靶向激酶的疗法约占癌症药物发现工作的 50%。在实验室探索人类激酶生物化学和生物物理学的能力对于设计选择性抑制剂和研究耐药性至关重要。细菌表达系统在简单性和成本效益方面优于昆虫或哺乳动物细胞,但历史上一直在人类激酶表达方面遇到困难。在发现磷酸酶共表达在细菌中产生高产量的 Src 和 Abl 激酶结构域后,我们生成了一个包含 52 个 His 标记的人激酶结构域构建体的文库,这些构建体在利用磷酸酶共表达的自动化细菌表达系统中表达超过 2 μg/mL 的培养物(YopH 代表 Tyr 激酶,lambda 代表 Ser/Thr 激酶)。在这里,我们报告了一种结构生物信息学方法,用于识别先前在细菌中表达的激酶结构域构建体,并且可能在我们的方案中表达良好,实验证明我们的简单构建体选择策略在 Abl 的 84 个潜在激酶域边界测试中选择具有良好表达产量的构建体,并通过 96 种人类激酶构建体的高通量表达筛选得到。使用基于荧光的热稳定性测定和荧光 ATP 竞争性抑制剂,我们表明表达最高的激酶被折叠并具有良好形成的 ATP 结合位点。我们还证明,这些构建体可以通过表达一组 48 个 Src 和 46 个 Abl 突变来表征临床突变。野生型激酶构建体库可通过 Addgene 公开获得。
更新日期:2018-07-13
中文翻译:
用于自动化细菌表达的人激酶结构域构建的开放库
激酶在细胞信号传导中发挥着关键作用,并且在许多疾病中失调,例如癌症、糖尿病和神经退行性疾病。目前,靶向激酶的疗法约占癌症药物发现工作的 50%。在实验室探索人类激酶生物化学和生物物理学的能力对于设计选择性抑制剂和研究耐药性至关重要。细菌表达系统在简单性和成本效益方面优于昆虫或哺乳动物细胞,但历史上一直在人类激酶表达方面遇到困难。在发现磷酸酶共表达在细菌中产生高产量的 Src 和 Abl 激酶结构域后,我们生成了一个包含 52 个 His 标记的人激酶结构域构建体的文库,这些构建体在利用磷酸酶共表达的自动化细菌表达系统中表达超过 2 μg/mL 的培养物(YopH 代表 Tyr 激酶,lambda 代表 Ser/Thr 激酶)。在这里,我们报告了一种结构生物信息学方法,用于识别先前在细菌中表达的激酶结构域构建体,并且可能在我们的方案中表达良好,实验证明我们的简单构建体选择策略在 Abl 的 84 个潜在激酶域边界测试中选择具有良好表达产量的构建体,并通过 96 种人类激酶构建体的高通量表达筛选得到。使用基于荧光的热稳定性测定和荧光 ATP 竞争性抑制剂,我们表明表达最高的激酶被折叠并具有良好形成的 ATP 结合位点。我们还证明,这些构建体可以通过表达一组 48 个 Src 和 46 个 Abl 突变来表征临床突变。野生型激酶构建体库可通过 Addgene 公开获得。