Lysosomal lipid-binding proteins: purification

From LipidomicsWiki

Purification of recombinant his-tagged GM2-Activatorprotein from baculovirus-expression-system

Endosomal / lysosomal Lipid transfer-proteins are required for transport and processing of lipids within the endosomal / lysosomal system. The GM2-activator protein and the saposins A-D act in part as cofactors of hydrolytic enzymes. The GM2-activator is required for the degradation of ganglioside GM2 by beta-hexosaminidase A. Its deficiency leads to the AB-variant of GM2-gangliosidoses.

Liposomal assays of GM2-degradation by beta-hexosamindase A require the presence of the GM2-activator. This and other lysosomal lipid transfer-proteins can be prepared in pure form as glycoproteins from eukaryotic expression systems.

Representative purification protocol for recombinant GM2-activator

1. Treatment of supernatant of baculovirus-infected insect cells (Spodoptera frugiperda)
- centrifuge insect cells at 5000 g for 15 min, harvest supernatant
- adjust supernatant to pH 3.8
- centrifuge at 9000 g for 15 min
- transfer supernatant to a cation exchanger (Poros 20HS, 2 mL/min)

2. Cation exchange chromatography

- pre-equilibrate column with 10 column-volume binding-buffer (50 mM NaH₂P0₄, pH 4.0)
- transfer supernatant to column (2 mL/min)
- wash column with 10 column-volume binding-buffer (50 mM NaH₂P0₄, pH 4.0)
- elute the protein by a salt gradient (10 column-volumes from 0 M NaCl to 1M NaCl in 50 mM NaH₂P0₄, pH 4.0), collect eluate in 1 mL-fractions
- combine protein-containing fractions for purification by Ni-NTA

3. Immobilized Metal Ion Affinity Chromatography (nickel-nitrilotriacetic acid, NiNTA)

Column: prepared from NiNTA (Qiagen, 1018240); column volume 5 mL

NiNTA-Buffer: 

a) 10x NiNTA Buffer pH 8.0

500 mM Na₂HP0₄ x 2H₂0 a 89.00 g/l
3 M NaCl  a 175.32 g/l

adjust to pH 8.0 ( H₃PO₄ / NaOH)

b) 10x NiNTA Buffer pH 3.8

500 mM NaH₂P0₄ x 2H₂0 a 89.00 g/l
3 M NaCl a 175.32 g/l

adjust to pH 3.8 ( H₃PO₄ / 10x NiNTA-Puffer pH 8)

c) 10x NiNTA Buffer pH 7.0 and pH 6.0

by mixing of buffers pH 8 and pH 4

- pre-equilibrate the column with 10 column-volume NiNTA-Buffer pH 8
- mix the combined GM2AP-fractions with 1/10 volume of 10x NiNTA-Buffer, adjust pH to 8.0
- transfer the probes to the column (1mL/min)
- wash column with 10 column-volume of 1x NiNTA-Buffer pH 8, pH 7 and pH 6
- elute protein with 10 column-volume of 1x NiNTA-Buffer pH 3.8
- collect eluate in 1mL fractions
- Analyse eluted protein by 12.5% SDS-PAGE and silver staining

4. Source

S. Anheuser, Dissertation Universität Bonn, in preparation. Modified from <ref>Wendeler M, Hoernschemeyer J, John M, Werth N, Schoeniger M, Lemm T, Hartmann R, Kessler H, Sandhoff K., Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry. Protein Expr Purif, 2003. 27, 259-66</ref>