Abstract：
A previous study reported the use of a biosensing technique based on surface plasmon resonance (SPR) for the ligand binding detection of peroxisome proliferator activator receptor gamma (PPARγ). This detection was designed based on the structural properties of PPARγ. Because of cross-linked protein inactivation and the low molecular weight of conventional ligands, direct ligand binding detection based on SPR has low stability and repeatability. In this study, we report an indirect response methodology based on SPR technology in which anti-His CM5 chip binds fresh PPARγ every cycle, resulting in more stable detection. We developed a remarkable improvement in ligand-protein binding detectability in vitro by introducing two coregulator-related polypeptides into this system. In parallel, a systematic indirect response methodology can reflect the interaction relationship between ligands and proteins to some extent by detecting the changes in SA-SRC1 and GST-NCOR2 binding to PPARγ. Rosiglitazone, a PPARγ agonist with strong affinity, is a potent insulin-sensitizing agent. Some ligands may be competitively exerted at the same sites of PPARγ (binding rosiglitazone). We demonstrated using indirect response methodology that selective PPARγ modulator (SPPARM) candidates of PPARγ can be found by competing for the binding of the rosiglitazone site on PPARγ, although they may have no effect on polypeptides and PPARγ binding.
摘要：
先前的研究报道了基于表面等离子体共振（SPR）的生物传感技术用于过氧化物酶体增殖物激活物受体γ（PPARγ）的配体结合检测。该检测是基于PPARγ的结构特性设计的。由于交联蛋白失活和常规配体的低分子量,基于SPR的直接配体结合检测具有较低的稳定性和重复性。在这项研究中,我们报告了一种基于SPR技术的间接反应方法，其中抗HisCM5芯片每个周期结合新鲜的PPARγ，导致更稳定的检测。通过向该系统中引入两个与共调节因子相关的多肽，我们在体外对配体-蛋白质结合的可检测性进行了显着改善。并行,系统的间接反应方法可以通过检测SA-SRC1和GST-NCOR2与PPARγ结合的变化，在一定程度上反映配体与蛋白质之间的相互作用关系。罗格列酮,一种具有强亲和力的PPARγ激动剂，是一种有效的胰岛素增敏剂。一些配体可以竞争性地施加在PPARγ的相同位点(结合罗格列酮)。我们使用间接反应方法证明，通过竞争PPARγ上的罗格列酮位点的结合，可以找到PPARγ的选择性PPARγ调节剂（SPPRM）候选物。尽管它们可能对多肽和PPARγ结合没有影响。
