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首页> 外文期刊>Protein Expression and Purification >Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae
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Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae

机译：酿酒酵母中的第二个NMN腺苷酸转移酶基因的鉴定和表征

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摘要

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD(+). On the basis of a remarkable structural similarity with previously described Saccharomyces cerevisiae NMNAT (yNMNAT-1), the YGR010-encoded protein was identified as a second isoform of yeast NMNAT (yNMNAT-2). The YGR010 gene was isolated, cloned into a T7-based vector, and successfully expressed in Escherichia coli BL21 cells, yielding high level of NMN adenylyltransferase activity. The purification procedure reported in this paper, consisting of two chromatographic steps, allowed the isolation of 3 mg of electrophoretically homogeneous yNMNAT-2 from 1 liter of E coli culture. Under SDS/PAGE, the recombinant protein resulted in a single polypeptide of 46 kDa, in agreement with the molecular mass of the hypothetical protein encoded by YGR010 gene. The N-terminal sequence of the purified recombinant yNMNAT-2 exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant yNMNAT-2 are reported and compared with those already known for yNMNAT-1. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 38]
机译：烟酰胺单核苷酸（NMN）腺苷酸转移酶（NMNAT）（EC 2.7.7.1）催化ATP的腺苷基部分向NMN的转移以形成NAD（+）。基于与先前描述的酿酒酵母NMNAT（yNMNAT-1）的显着结构相似性，鉴定出YGR010编码的蛋白是酵母NMNAT的第二同工型（yNMNAT-2）。分离YGR010基因，将其克隆到基于T7的载体中，并在大肠杆菌BL21细胞中成功表达，从而产生高水平的NMN腺苷酸转移酶活性。本文报道的纯化程序包括两个色谱步骤，可从1升大肠杆菌培养物中分离出3 mg电泳均质的yNMNAT-2。在SDS / PAGE下，重组蛋白产生单个46kDa的多肽，与YGR010基因编码的假定蛋白的分子量一致。纯化的重组yNMNAT-2的N端序列与预测的序列完全对应。报告了重组yNMNAT-2的分子和动力学特性，并将其与已知的yNMNAT-1进行了比较。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：38]

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