人FAD合成酶同工型2在大肠杆菌中的过表达、纯化及特性分析

Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.

作者信息

Galluccio Michele, Brizio Carmen, Torchetti Enza Maria, Ferranti Pasquale, Gianazza Elisabetta, Indiveri Cesare, Barile Maria

机构信息

Dipartimento di Biologia Cellulare, Università della Calabria, Via P. Bucci 4c, I-87036 Arcavacata di Rende, Italy.

出版信息

Protein Expr Purif. 2007 Mar;52(1):175-81. doi: 10.1016/j.pep.2006.09.002. Epub 2006 Sep 12.

DOI:10.1016/j.pep.2006.09.002

PMID:17049878

Abstract

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.

摘要

黄素腺嘌呤二核苷酸合成酶（FADS）（EC 2.7.7.2）是将核黄素转化为氧化还原辅因子黄素腺嘌呤二核苷酸（FAD）的代谢途径中的关键酶。人FADS同工型2（hFADS2）是FLAD1基因的产物，作为T7标签蛋白在大肠杆菌中过表达，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析进行鉴定。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）计算，其分子量约为55 kDa。表达的蛋白占从细胞培养物中提取的总蛋白的40%以上；通过用Triton X-100处理不溶性细胞部分，以可溶且近乎纯的形式回收了其中的10%。如分光光度法测定所示，hFADS2具有FADS活性，并且对氯化镁有严格要求。纯化的重组同工型2的催化常数（kcat）为3.6×10⁻³ s⁻¹，对黄素单核苷酸（FMN）的米氏常数（KM）约为0.4 μM。hFADS2同工型的表达为该酶的结构研究以及基于细菌和人类酶功能差异的抗生素设计开辟了新的前景。