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, Galactofuranosyltransferase, Aspergillus, Protein purification
Materials and Reagents
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Aspergillus nidulans expressing 3x FLAG-tagged GfsA (Komachi et al., 2013)
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3x FLAG-peptide (Sigma-Aldrich, catalog number: F4799 )
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ANTI-FLAG M2-agarose produced from mouse (Sigma-Aldrich, catalog number: A2220 )
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Mouse IgG-agarose (Sigma-Aldrich, catalog number: A0919 )
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2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (Dojindo Molecular Technologies, catalog number: GB10 )
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Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
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Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
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Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
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Manganese (II) chloride tetrahydrate (MnCl2) (Wako Pure Chemical Industries, catalog number: 133-00725 )
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Glycerol (Wako Pure Chemical Industries, catalog number: 075-00611 )
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3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO) (Dojindo Molecular Technologies, catalog number: C020 )
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CompleteTM protease inhibitor cocktail tablets (EDTA-free) (Roche Diagnostics, catalog number: 1873580 )
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Liquid nitrogen
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Buffer A (see Recipes)
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Minimal medium (MM) (see Recipes)
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Hutner's trace elements (see Recipes)
Equipment
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Spreader
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500-ml Sakaguchi flasks
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Mortar and pestle
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Aspirator
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Centrifuge with an angle rotor
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Centrifuge with a swing rotor
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Ultracentrifuge
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Spatula
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15-ml plastic centrifuge tube (e.g., Greiner Bio-One GmbH)
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4 °C incubator
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30 °C incubator
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Rotator (e.g., TAITEC)
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Filter paper (Munktell & Filtrak GmbH, catalog number: 113053 )
Procedure
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Streak Aspergillus nidulans conidia from frozen stock onto Minimal medium (MM) plate and cultivate for 3 days at 30 °C.
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Collect the formed conidia with a spreader.
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Spread Aspergillus nidulans conidia (1 x 105) onto MM plates and cultivate for 3 days at 30 °C.
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Inoculate the collected conidia (2 x 107) into 100 ml of MM in 500-ml Sakaguchi flasks.
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Shake the flasks at 126 rpm at 30 °C for 24 h.
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Collect the mycelial cells by paper filtration.
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Wash the cells twice with approximately 30 ml of distilled water.
Note: Cells can easily be crushed by wringing wet cells out to dry using a scoopula after this step as much as possible.
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Grind cells (25 g of wet cells) into a fine powder in liquid nitrogen using a mortar and pestle.
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Resuspend the lysed cells in 100 ml of buffer A containing CompleteTM protease inhibitor cocktail (EDTA-free).
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Remove cell debris by centrifugation with an angle rotor at 10,000 x g for 10 min.
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Centrifuge the supernatant at 100,000 x g for 45 min using an ultracentrifuge.
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Resuspend the resultant pellet in 10 ml of buffer A containing 0.5% CHAPSO using a spatula.
Note: Pilot experiments are needed to determine the suitable conditions under which the detergents solubilize the target protein.
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Gently mix the sample for 1 h using a rotator to obtain solubilized membrane proteins.
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Centrifuge the sample at 100,000 x g for 30 min using an ultracentrifuge.
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Collect the supernatant (approximately 10 ml) into a 15-ml plastic centrifuge tube.
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Add mouse-IgG-agarose (100 µl) to the supernatant and gently shake the mixture for 1 h (Video 1).
Video 1. Shaking the mixture using a rotator
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Remove the mouse-IgG-agarose by centrifugation with a swing rotor at 1,400 x g for 10 min.
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Add 200 µl anti-FLAG M2 affinity gel to the supernatant and gently shake the resultant mixture for 1 h.
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Collect the Anti-FLAG M2 affinity gel by centrifugation with a swing rotor at 1,400 x g for 10 min.
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Gently remove the supernatant with an aspirator.
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Resuspend the resultant agarose with 15 ml of buffer A containing 0.1% CHAPSO.
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Repeat steps 18-20 five times.
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Elute GfsA protein with 20 µl of buffer A with 0.1% CHAPSO containing 0.5 µg/µl 3x FLAG peptide.

Figure 1. Purification of GfsA-3xFLAG protein. A total of 0.5 mg (silver staining) proteins were separated by 5%-20% SDS-PAGE, and were then assayed by silver staining. GfsA-3xFLAG was detected as a 67 kDa protein. Asterisk indicates a degradation product or insufficiently N-glycosylated product of GfsA-3xFLAG.
Notes
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Perform all manipulations on ice or at 4 °C.
Recipes
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Buffer A (1 L)
Compounds
|
Amounts
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HEPES
|
11.9 g
|
NaCl
|
5.84 g
|
KCl
|
2.24 g
|
MnCl2.4H2O
|
0.2 g
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Glycerol
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50 g
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Add water to bring the final solution to 1 L total volume
Filter sterilize the solution using a 0.45 μm filter
Stored at 4 °C
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Minimal medium (1 L)
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
|
Adjust pH to 6.8 using NaOH
Add water to bring the final solution to 1 L total volume
Autoclave for 20 min
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Hutner's trace elements
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
|
Adjust the pH value to 6.5-6.8 using KOH
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
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Komachi, Y., Hatakeyama, S., Motomatsu, H., Futagami, T., Kizjakina, K., Sobrado, P., Ekino, K., Takegawa, K., Goto, M., Nomura, Y. and Oka, T. (2013). GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol Microbiol 90(5): 1054-1073.
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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.