Published: Vol 4, Iss 17, Sep 5, 2014 DOI: 10.21769/BioProtoc.1222 Views: 8998
Reviewed by: Kanika GeraFanglian He
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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: GalactofuranoseMaterials and Reagents
Equipment
Procedure
Notes
Recipes
Compounds | Amounts |
HEPES | 11.9 g |
NaCl | 5.84 g |
KCl | 2.24 g |
MnCl2.4H2O | 0.2 g |
Glycerol | 50 g |
Compounds | Amounts |
NaNO3 | 6.0 g |
KCl | 0.52 g |
MgSO4.7H20 | 0.52 g |
KH2PO4 | 1.52 g |
Glucose | 10.0 g |
Hutner's trace elements | 2 ml |
Compounds | Amounts |
H2O (60 °C) | 100 ml |
ZnSO4.7H2O | 2.2 g |
H3BO3 | 1.1 g |
MnCl2.4H2O | 0.5 g |
FeSO4.7H2O | 0.5 g |
CoCl2.6H2O | 0.16 g |
CuSO4.5H2O | 0.16 g |
(NH4) 6Mo7O24.4H2O | 0.11 g |
EDTA | 5.0 g |
Acknowledgments
This protocol was adapted from the previously published paper Komachi et al., 2013. The work was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society of the Promotion of Science (JSPS) (21780313, 23780350 and 26450106) (to T.O.).
References
Article Information
Copyright
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
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.
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Expression
Biochemistry > Protein > Labeling
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