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Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist.
Protein Engineering, Design and Selection ( IF 2.4 ) Pub Date : 2017-12-28 , DOI: 10.1093/protein/gzx062
Ewa Oclon 1, 2 , Gili Solomon 3 , Zvi Hayouka 3 , Tomer Meir Salame 4 , Vincent Goffin 5 , Arieh Gertler 3
Affiliation  

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

中文翻译:

用于人类催乳激素研究的新型试剂:催乳素受体胞外域,非聚乙二醇化和聚乙二醇化催乳素和催乳素受体拮抗剂的大规模制备和表征。

为了提供用于体外和体内催乳激素(PRL)研究的新工具,用于大规模制备未标记人PRL(hPRL),hPRL拮抗剂(del 1-9-G129R hPRL)的新方案,该新试剂可作为人类泌乳素的纯拮抗剂证明了hPRL与hPRL受体胞外域(hPRLR-ECD)和hPRLR-ECD结合。通过使用生物素化的hPRL作为配体和hPRLR-ECD作为受体的竞争性非放射性结合试验,通过与hPRLR-ECD形成稳定的1:1复合物,证明了del 1-9-G129R hPRL与hPRLR-ECD的相互作用。使用尺寸排阻色谱法和表面等离振子共振方法的非变性条件。在所有三种类型的实验中,del 1-9-G129R hPRL的相互作用与未修饰的hPRL的相互作用相等。Del 1-9-G129R hPRL抑制了hPRL诱导的稳定表达hPRLR的Baf / LP细胞的增殖。总体而言,通过本文所述方案制备的del 1-9-G129R hPRL的生物学特性与使用原始研究中报道的方案制备的拮抗剂的生物学特性相似。但是,新描述的方案将产量提高了6倍以上。为了提供持久的hPRL作为体内实验所需的新试剂,我们制备了其单聚乙二醇化类似物,并发现聚乙二醇化在同源体外测定中降低了其生物活性。由于其未来用途将需要开发具有高度提高的亲和力的PRL拮抗剂,因此在酵母细胞表面表达了del 1-9-G129R hPRL。它保留了对hPRLR-ECD的结合能力,
更新日期:2019-11-01
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