绵羊COX-1同工酶的生物生产

Q4 Pharmacology, Toxicology and Pharmaceutics
Morena Miciaccia, M. Iaselli, S. Ferorelli, P. L. Polosa, M. Perrone, A. Scilimati
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引用次数: 0

摘要

最近的研究结果揭示了环氧合酶-1和-2 (COX-1和COX-2)在以炎症为主要早期阶段的人类疾病,如癌症和神经退行性疾病中的关键作用。cox是非甾体抗炎药的主要靶点,它催化花生四烯酸双氧合生成前列腺素PGH2,然后通过组织特异性异构酶转化为前列腺素、血栓素和前列腺环素。为了研究COX-1的结构和功能,以及体外疾病生化途径的研究,需要大量的COX-1纯结果。用杆状病毒感染夜蛾细胞,发现杆状病毒是一个高效的表达系统,可获得大量的羊COX-1。改进了蛋白质在非离子洗涤剂存在下的溶解时间,并介绍了第二纯化步骤。对先前报道的纯重组oCOX-1生产和分离方法的改进已经实现,导致每次纯化时感染细胞的起始体积降低,细胞密度增加,每个细胞的病毒颗粒数量增加,感染周期缩短。深入阐述了重组oCOX-1表达和纯化的方案,以获得1 mg/L的蛋白。优化后的工艺也适用于其他膜蛋白的制备,需要对增溶步骤进行改进,以获得高浓度的膜蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Ovine COX-1 isoenzyme bio-production
Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 results is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, and of the number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein. The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins.
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来源期刊
Current Enzyme Inhibition
Current Enzyme Inhibition Pharmacology, Toxicology and Pharmaceutics-Drug Discovery
CiteScore
1.30
自引率
0.00%
发文量
30
期刊介绍: Current Enzyme Inhibition aims to publish all the latest and outstanding developments in enzyme inhibition studies with regards to the mechanisms of inhibitory processes of enzymes, recognition of active sites, and the discovery of agonists and antagonists, leading to the design and development of new drugs of significant therapeutic value. Each issue contains a series of timely, in-depth reviews written by leaders in the field, covering a range of enzymes that can be exploited for drug development. Current Enzyme Inhibition is an essential journal for every pharmaceutical and medicinal chemist who wishes to have up-to-date knowledge about each and every development in the study of enzyme inhibition.
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