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Author:
Date:
2014-09-05
[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAG-tagged GfsA produced a ...
[摘要] GfsA是涉及O - 聚糖的生物合成的真菌β-半乳糖呋喃糖基转移酶。 为了研究GfsA的酶功能，我们尝试从两种异源宿主生物体获得该酶的重组蛋白。 然而，GfsA不能在大肠杆菌（大肠杆菌）或酿酒酵母（Saccharomyces cerevisiae）中表达为重组蛋白质（ cerevisiae ）。 因此，我们决定使用构巢曲霉（ A。nidulans ）作为宿主生物体，并产生使用染色体标签表达3×FLAG标记的GfsA的菌株。 为了证实其表达，从标记的菌株制备溶解的蛋白并用抗FLAG抗体分析。 表达3×FLAG标记的GfsA的菌株产生质量为约67kDa的功能性蛋白质。 该手稿中描述的方法允许纯化在A中表达的GfsA-3xFLAG蛋白。 构巢细胞。
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