Diseño y producción de diversas proteínas fusión de la nicotinamida/nicotinato mononucleótido adenilil transferasa (NMNAT) de Plasmodium falciparum

Pub Date : 2017-09-01 DOI:10.15446/REV.COLOMB.QUIM.V46N3.63492

C. Clavijo, Nicolás Forero Baena, Marializ Magaly Ramírez Hernández

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引用次数: 1

Abstract

Recombinant proteins have become useful tools in biochemistry research. During their production, however, inclusion bodies (IB) appear, on the one hand, due to the high expression rate from the recombinant plasmids, which have high efficiency promoters, and, on the other hand, intrinsic characteristics of the expressed protein. Furhtermore, the nicotinamide/nicotinate mononucleotide adenilyl transferase (NMNAT) is a central protein in NAD(H)+ biosynthesis, an essential cofactor in cell metabolism, and in protozoon parasite has been studied. To study the NMNAT protein of these parasites, their recombinant version in E. coli has been expressed, getting a great quantity of IB as a by-product. To increase the solubility of the protein, the coding sequence of the NMNAT enzyme of Plasmodium falciparum was cloned in different expression plasmids which were subsequently transformed into E. coli BL21(DE3) expression strain. The solubility of the recombinant proteins was assessed and the one with the highest presence in the soluble fraction was subsequently purified and its enzyme activity was determined. The recombinant protein with a MBP (maltose-binding protein) tag showed an increased solubility and purity.

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重组蛋白已成为生物化学研究的有用工具。然而，在其生产过程中，包含体(IB)的出现，一方面是由于重组质粒具有高效启动子的高表达率，另一方面是由于所表达蛋白的固有特性。烟酰胺/烟酸单核苷酸腺苷转移酶(NMNAT)是NAD(H)+生物合成的中心蛋白，是细胞代谢的重要辅助因子，在原虫寄生虫中也有研究。为了研究这些寄生虫的NMNAT蛋白，我们在大肠杆菌中表达了它们的重组蛋白，得到了大量的IB作为副产物。为了提高该蛋白的溶解度，将恶性疟原虫NMNAT酶的编码序列克隆到不同表达质粒中，转化到大肠杆菌BL21(DE3)表达菌株中。评估了重组蛋白的溶解度，随后纯化了可溶性部分中存在最高的重组蛋白，并测定了其酶活性。带有MBP(麦芽糖结合蛋白)标签的重组蛋白显示出更高的溶解度和纯度。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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