Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans
构巢曲霉中表达的GfsA-3xFLAG 蛋白的纯化
发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1222 浏览次数: 9216
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
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 functional protein with a mass of approximately 67 kDa. The method described in this manuscript allows purification of the GfsA-3xFLAG protein as expressed in A. nidulans cells.
Keywords: Galactofuranose (呋喃)
Galactofuranosyltransferase (galactofuranosyltransferase)
Aspergillus (曲霉)
Protein purification (蛋白纯化)
Materials and Reagents
- Aspergillus nidulans expressing 3x FLAG-tagged GfsA (Komachi et al., 2013)
- 3x FLAG-peptide (Sigma-Aldrich, catalog number: F4799 )
- ANTI-FLAG M2-agarose produced from mouse (Sigma-Aldrich, catalog number: A2220 )
- Mouse IgG-agarose (Sigma-Aldrich, catalog number: A0919 )
- 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (Dojindo Molecular Technologies, catalog number: GB10 )
- Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
- Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
- Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
- Manganese (II) chloride tetrahydrate (MnCl2) (Wako Pure Chemical Industries, catalog number: 133-00725 )
- Glycerol (Wako Pure Chemical Industries, catalog number: 075-00611 )
- 3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO) (Dojindo Molecular Technologies, catalog number: C020 )
- CompleteTM protease inhibitor cocktail tablets (EDTA-free) (Roche Diagnostics, catalog number: 1873580 )
- Liquid nitrogen
- Buffer A (see Recipes)
- Minimal medium (MM) (see Recipes)
- Hutner's trace elements (see Recipes)
Equipment
- Spreader
- 500-ml Sakaguchi flasks
- Mortar and pestle
- Aspirator
- Centrifuge with an angle rotor
- Centrifuge with a swing rotor
- Ultracentrifuge
- Spatula
- 15-ml plastic centrifuge tube (e.g., Greiner Bio-One GmbH)
- 4 °C incubator
- 30 °C incubator
- Rotator (e.g., TAITEC)
- Filter paper (Munktell & Filtrak GmbH, catalog number: 113053 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Oka, T., Katafuchi, Y., Fukuda, K., Ekino, K., Goto, M. and Nomura, Y. (2014). Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans. Bio-protocol 4(17): e1222. DOI: 10.21769/BioProtoc.1222.
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分类
微生物学 > 微生物生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 表达
生物化学 > 蛋白质 > 标记
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