Bertinetti Daniela, Schweinsberg Sonja, Hanke Susanne E, Schwede Frank, Bertinetti Oliver, Drewianka Stephan, Genieser Hans-Gottfried, Herberg Friedrich W
Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
Biolog Life Science Institute, Flughafendamm 9a, P.O. Box 107125, Bremen, Germany.
BMC Chem Biol. 2009 Feb 12;9:3. doi: 10.1186/1472-6769-9-3.
In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins.
Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources.
In summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.
在真核细胞中，环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）是信号转导中的关键酶，是第二信使cAMP的主要作用靶点。在此，我们描述了经过特殊设计的cAMP类似物的设计、合成及特性，这些类似物可用作亲和富集和纯化的工具，以及用于基于蛋白质组学的cAMP结合蛋白分析。
基于cAMP的硫代磷酸酯衍生物开发了两组化学结合剂，Sp-cAMPS和Rp-cAMPS分别作为cAMP激动剂和拮抗剂。通过直接表面等离子体共振（SPR）分析测试了这些化合物与PKA R亚基和全酶的结合特性。此外，这些类似物用于亲和纯化方法，以分析它们对PKA R亚基所代表的cAMP结合蛋白的富集和改善的结合及洗脱特性。通过SPR测定，所有测试的Sp类似物都为亲和色谱提供了有价值的工具。然而，Sp-8-AEA-cAMPS表现出：（i）卓越的富集特性，同时对粗细胞裂解物中的其他蛋白质保持低非特异性结合；（ii）允许温和的洗脱条件；（iii）能够在8小时内以毫克量高效纯化所有四种活性PKA R亚基同工型。在化学蛋白质组学方法中，两组结合剂，即Rp-和Sp-cAMPS衍生物均可使用。虽然Sp-8-AEA-cAMPS优先结合游离R亚基，但具有拮抗剂特性的Rp-AHDAA-cAMPS不仅与游离PKA R亚基结合，还与重组和内源性来源的完整PKA全酶结合。
总之，所有测试的cAMP类似物作为亲和试剂在各自的应用中都很有用，可增强cAMP结合蛋白的纯化。Sp-8-AEA-cAMPS被认为是最有效的类似物，因为Sp-8-AHA-cAMPS和Sp-2-AHA-cAMPS表现出从基质中不完全洗脱，以及保留大量结合的蛋白质污染物。此外，可以证明基于Rp-8-AHDAA-cAMPS的亲和树脂为化学蛋白质组学方法提供了有价值的工具。
