Purification of Lysosomal Enzymes from Conditioned Media

Culture media were centrifuged at 500 × g for 20 min and filtered (0.45 μm). For AGA, 20% v/v of conditioning buffer (70 mM Tris-HCl, pH 7.0) was added to the media and loaded on column packed with Q-FastFlow Sepharose (GE Healthcare) pre-equilibrated with 5 column volume (CV) equilibration buffer (20 mM Tris-HCl, 20 mM sodium acetate, 70 mM sodium chloride, pH 6.8). After washing the column with 6 CV of wash buffer (20 mM Tris-HCl, 20 mM sodium acetate, 70 mM sodium chloride, pH 6.8), the enzyme was one-step eluted with elution buffer (25 mM sodium acetate, 250 mM NaCl, pH 4.5) into a tube containing 300 mM sodium phosphate (pH 7.3). The eluates were diluted with 50% v/v of 4 M (NH₄)₂SO₄ and further loaded on a Phenyl-Sepharose Fast Flow (high substitution) column (GE Healthcare). After washing and equilibrating the column with 5 CV of 2 M (NH₄)₂SO₄, 20 mM Tris-HCl, pH 7.0, the enzyme was eluted with elution buffer in gradient (2–0 M (NH₄)₂SO₄, 20 mM Tris-HCl, pH 7.0). For GUSB, medium was diluted 3-fold with conditioning buffer (10 mM Tris-HCl, 1 mM β-glycerophosphate, pH 8.0) and loaded on HiTrap DEAE Sepharose Fast Flow column (GE Healthcare) pre-equilibrated with 2 CV conditioning buffer. After washing the column with wash buffer (10 mM Tris-HCl, 1 mM β-glycerophosphate, 50 mM NaCl, pH 8.0), the enzyme was eluted in elution buffer (10 mM Tris-HCl, 1 mM β-glycerophosphate, 300 mM NaCl, pH 8.0). Eluates were diluted in 3-fold volume of conditioning buffer (10 mM Tris-HCl, 1 mM β-glycerophosphate, pH 8.0) and loaded on Mono-Q column (GE Healthcare) and eluted with elution buffer in gradient (0–1 M NaCl, 10 mM Tris-HCl, 1 mM β-glycerophosphate, pH 8.0). For CTSD, medium was diluted 3:1 (v/v) with conditioning buffer (100 mM Tris, 40 mM imidazole 1.2 M NaCl, pH 8.0) and loaded on a 1 mL packed Histrap column (GE Healthcare) pre-equilibrated with 5 CV of conditioning buffer (25 mM Tris, 10 mM imidazole, 300 mM NaCl, pH 8.0). The column was washed with 5CV of conditioning buffer and the enzyme was eluted with 4CV of elution buffer (250 mM imidazole, 25 mM Tris, 300 mM NaCl, pH8.0). For TPP1, medium was diluted 3:1 (v/v) with conditioning buffer (20 mM Tris–HCl, pH 7.6) and loaded on DEAE-Sepharose Fast Flow column pre-equilibrated with 5 CV of conditioning buffer. The enzyme was eluted stepwise with 25, 100, 200 mM, 400 mM NaCl in 2 CV of conditioning buffer. Eluates were diluted with 3X volume of conditioning buffer and thereafter loaded onto Mono-Q column and eluted with gradient NaCl (0-1 M NaCl, 20 mM Tris–HCl, pH 7.6). For GAA, medium was dialysed over night and loaded on a DEAE-Sepharose Fast Flow column pre-equilibrated with 5 CV of 25 mM MES, pH 6.5. The enzyme was eluted in one step with 200 mM NaCl in 2 CV of 25 mM MES, pH 6.5. Eluates were adjusted to 1 M (NH₄)₂SO₄ and loaded on a Phenyl-Sepharose Fast Flow column pre-equilibrated with 25 mM MES, 1 M (NH₄)₂SO_4, pH 6,5. The enzyme was eluted one step at 50 mM (NH₄)₂SO₄ in 25 mM MES. The eluates were buffer exchanged and further loaded on Mono-Q column and eluted in gradient in 0–1 M NaCl, 25 mM MES, pH 6.5. For IDS, medium was dialysed overnight and loaded on a DEAE-Sepharose Fast Flow column pre-equilibrated with 5 CV of 25 mM MES, pH 6.5. The enzyme was eluted one step with 200 mM NaCl in 2 CV of 25 mM MES, pH 6.5. Eluates were adjusted to 2 M NaCl and loaded on a Phenyl-Sepharose Fast Flow column pre-equilibrated with 25 mM MES, 2 M NaCl, pH 6,5. The enzyme was eluted one step at 150 mM NaCl in 25 mM MES, pH 6,5. The eluates were buffer exchanged and further loaded on Mono-Q column and eluted in gradient in 0–1 M NaCl, 25 mM MES, pH 6.5.

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Chen Y.H., Tian W., Yasuda M., Ye Z., Song M., Mandel U., Kristensen C., Povolo L., Marques A.R., Čaval T., Heck A.J., Sampaio J.L., Johannes L., Tsukimura T., Desnick R., Vakhrushev S.Y., Yang Z, & Clausen H. (2023). A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution. Frontiers in Bioengineering and Biotechnology, 11, 1128371.

Publication 2023

Buffer Ctsd Deae Enzyme Imidazole Mono q Nacl Phenyl sepharose Sepharose Sodium acetate Sodium phosphate Tris β glycerophosphate

Corresponding Organization : Novo Nordisk (Denmark)

Other organizations : Technical University of Denmark, Novo Nordisk Foundation, Kiel University, Utrecht University, Institut Curie, Université Paris Sciences et Lettres, Centre National de la Recherche Scientifique, Inserm, Meiji Pharmaceutical University

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Variable analysis

independent variables

Centrifugation speed (500 × g)
Centrifugation time (20 min)
Filtration (0.45 μm)
Volume ratio of conditioning buffer to media (20% v/v)
Equilibration buffer composition (20 mM Tris-HCl, 20 mM sodium acetate, 70 mM sodium chloride, pH 6.8)
Wash buffer composition (20 mM Tris-HCl, 20 mM sodium acetate, 70 mM sodium chloride, pH 6.8)
Elution buffer composition (25 mM sodium acetate, 250 mM NaCl, pH 4.5)
Volume ratio of eluate to (NH4)2SO4 (50% v/v)
(NH4)2SO4 concentration in wash and equilibration buffer (2 M)
(NH4)2SO4 concentration gradient in elution buffer (2–0 M)
Conditioning buffer composition for GUSB (10 mM Tris-HCl, 1 mM β-glycerophosphate, pH 8.0)
Wash buffer composition for GUSB (10 mM Tris-HCl, 1 mM β-glycerophosphate, 50 mM NaCl, pH 8.0)
Elution buffer composition for GUSB (10 mM Tris-HCl, 1 mM β-glycerophosphate, 300 mM NaCl, pH 8.0)
NaCl concentration gradient in elution buffer for GUSB (0–1 M)
Conditioning buffer composition for CTSD (100 mM Tris, 40 mM imidazole 1.2 M NaCl, pH 8.0)
Wash buffer composition for CTSD (25 mM Tris, 10 mM imidazole, 300 mM NaCl, pH 8.0)
Elution buffer composition for CTSD (250 mM imidazole, 25 mM Tris, 300 mM NaCl, pH8.0)
Conditioning buffer composition for TPP1 (20 mM Tris–HCl, pH 7.6)
NaCl concentration steps in elution buffer for TPP1 (25, 100, 200, 400 mM)
NaCl concentration gradient in elution buffer for TPP1 (0-1 M NaCl)
Dialysis of medium for GAA
DEAE-Sepharose column equilibration buffer for GAA (25 mM MES, pH 6.5)
Elution buffer for GAA (200 mM NaCl in 25 mM MES, pH 6.5)
(NH4)2SO4 concentration in wash and equilibration buffer for Phenyl-Sepharose column for GAA (1 M)
Elution buffer for GAA from Phenyl-Sepharose column (50 mM (NH4)2SO4 in 25 mM MES)
NaCl concentration gradient in elution buffer for GAA from Mono-Q column (0–1 M NaCl)
Dialysis of medium for IDS
DEAE-Sepharose column equilibration buffer for IDS (25 mM MES, pH 6.5)
Elution buffer for IDS from DEAE-Sepharose column (200 mM NaCl in 25 mM MES, pH 6.5)
NaCl concentration in wash and equilibration buffer for Phenyl-Sepharose column for IDS (2 M)
Elution buffer for IDS from Phenyl-Sepharose column (150 mM NaCl in 25 mM MES, pH 6.5)
NaCl concentration gradient in elution buffer for IDS from Mono-Q column (0–1 M NaCl)

dependent variables

Enzyme activity or yield (not explicitly mentioned)

control variables

PH of buffers (Tris-HCl, sodium acetate, MES)
Temperature (not explicitly mentioned)

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