Assay of lysosome sialic acid transporter

Authors：

Category

Nucleotide sugar transporters

Protocol Name

Authors

Miyaji, Takaaki
Department of Genomics and Proteomics, Advanced Science Research Center, Okayama University

Omote, Hiroshi *
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University

Moriyama, Yoshinori
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University

*To whom correspondence should be addressed.

KeyWords

Sialic acid transport Purification Sialin Reconstitution

Reagents

●	Buffer A 20 mM Tris-HCl (pH 8.0), 0.1 M sodium acetate, 10% glycerol, 0.5 mM dithiothreitol, 10 μg/mL pepstatin A and 10 μg/mL leupeptin
●	Solubilization buffer 20 mM MOPS-Tris (pH 7.0), 10% glycerol, 2% octylglucoside, 10 μg/mL pepstatin A and 10 μg/mL leupeptin
●	Wash buffer 20 mM MOPS-Tris (pH 7.0), 5 mM imidazole, 20% glycerol and 1% octylglucoside
●	Reconstitution buffer 20 mM MOPS-Tris (pH 7.0), 0.1 M potassium acetate and 5 mM magnesium acetate, 0.5 mM dithiothreitol
●	Reaction mixture 20 mM MOPS-Tris (pH 7.0) or 40 mM MES (pH 5.6), 5 mM magnesium acetate, 4 mM KCl, 0.1 M potassium acetate

Instruments

●	Clean bench
●	Centrifuge
●	Ultracentrifuge
●	Sonicator

Methods

Purification

1)	Suspend infected insect cells (1–2 × 10⁸ cells) by buffer A and disrupt cells by sonication using a TOMY UD200 tip sonifier.	Comment 0

2)	Centrifuge cell lysates at 700 × g for 10 min to remove debris.	Comment 0

3)	Centrifuge supernatant at 160,000 × g for 1 h.	Comment 0

4)	Suspend the pellet (membrane fraction) in the solubilization buffer to give approximately 2 mg protein/mL.	Comment 0

5)	Centrifuge at 260,000 × g for 30 min to remove insolubilzed membrane.	Comment 0

6)	Add supernatant to 1 mL of Ni-NTA Superflow resin (Qiagen) and incubate for 4 h.	Comment 0

7)	Wash resin with 20 mL of wash buffer in a column.	Comment 0

8)	Elute sialin from the resin with 3 mL of the same buffer containing 60 mM imidazole.	Comment 0

Store eluate containing purified sialin at −80°C. Purified sialin is stable without loss of activity for at least a few months.

Comment 0

Reconstitution & Transport assay

1)	Mix 20 μg of sialin with liposomes (0.5 mg lipid^; see* Comment), freeze at −80°C and leave at this temperature for more than 10 min.	Comment 1

2)	Quickly thaw mixture by holding the sample tube in the hands and dilute 30-fold with reconstitution buffer.	Comment 0

3)	Sediment proteoliposomes by centrifugation at 200,000 × g for 1 h at 4°C and suspend in 0.2 mL of reconstitution buffer.	Comment 0

4)	Add proteoliposomes (0.5 μg total protein) into the reaction mixture containing 100 μM [6-³H] sialic acid (0.5 MBq/mmol) and incubate at 27°C.	Comment 0

5)	Withdraw 120 μL aliquots at the appropriate times and centrifuge through a Sephadex G-50 (fine) spin column at 760 × g for 2 min.	Comment 0

Measure radioactivity in the eluate.

Comment 0

Notes

Purification

All procedures should be carried out under 4°C.

Reconstitution & Transport assay

Reconstitution of purified recombinant sialin into liposomes is carried out by the freeze-thaw method.

Figure & Legends

Copyrights

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Date of registration：2015-12-17 11:18:06