Method for purifying a sulfatase protein
A technology of sulfatase and arylsulfatase, which is applied in the field of purifying sulfatase protein, can solve problems such as weak interactions, and achieve the effect of shortening time and reducing volume
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Embodiment 1
[0110] All chromatographic steps were performed at room temperature. Figure 11 A flowchart showing the purification method according to Example 1. Chromatographic equipment: Akta Explorer, or Akta Purifier 100, or Akta Pilot from GE Healthcare.
[0111] The harvest was clarified by adding 5M NaCl to a NaCl concentration of 0.5M. The pH was adjusted to 7.2-7.6 by adding 1M Tris.
[0112] Wash the chromatographic column filled with IMAC Sepharose (column The volume is 5ml, 30 or 200ml, the chromatographic column type is GE Healthcare's HiTrap or XK16 or XK50), and then 0.1-0.2M ZnCl is introduced first 2 (1CV) with Zn 2 + , followed by distilled water (1.5CV) and equilibrated with buffer A (2CV).
[0113] Load the harvest prepared as described above onto an equilibrated IMAC column at a flow rate of 50-150 cm / h with buffer A (5 CV), B - 20-50 mM Tris-HCl, 1-2 M NaCl, pH 7.4 -7.5 (10CV), C - 20-50 mM NaOAc, 100 mM NaCl, 0-10% i-PrOH, pH 6.4 (10CV), D - 20-50 mM NaOAc, 20-...
Embodiment 2
[0134] All chromatographic steps were performed at room temperature. Figure 13 A flowchart showing the purification method according to Example 2. Chromatographic equipment: Akta Explorer, or Akta Purifier 100, or Akta Pilot from GE Healthcare.
[0135] The harvest was clarified by adding 5M NaCl to a NaCl concentration of 0.5M. The pH was adjusted to 7.2-7.6 by adding 1M Tris.
[0136] Wash the chromatographic column filled with IMAC Sepharose ( The column volume is 5ml, 30 or 200ml, and the chromatographic column type is GE Healthcare's HiTrap or XK16 or XK50). Then pass through 0.1-0.2M ZnCl 2 (1CV) with Zn 2 + , followed by distilled water (1.5CV) and equilibrated with buffer A (2CV).
[0137] Load the harvest prepared as described above onto an equilibrated IMAC column at a flow rate of 50-150 cm / h with buffer A (5 CV), B - 20-50 mM Tris-HCl, 1-2 M NaCl, pH 7.4 -7.5 (10CV), C - 20-50 mM NaOAc, 100 mM NaCl, 0-10% i-PrOH, pH 6.4 (10CV), D - 20-50 mM NaOAc, 20-100 mM ...
Embodiment 3
[0153] All chromatographic steps were performed at room temperature. Figure 15 A flowchart showing the purification method according to Example 3. Chromatographic equipment: Akta Explorer, or Akta Purifier 100, or Akta Pilot from GE Healthcare.
[0154] The harvest was clarified by adding 5M NaCl to a NaCl concentration of 0.5M. The pH was adjusted to 7.2-7.6 by adding 1M Tris.
[0155] With distilled water (2CV), the Tris-HCl of buffer A-20-50mM, the NaCl of 0.5M, pH7.4-7.5-(2CV), the chromatographic column that distilled water (1.5CV) is filled with IMAC Sepharose ( The column volume is 5ml, 30 or 200ml, and the chromatographic column type is HiTrap or GE Healthcare's XK16 or XK50), and then pass through 0.1-0.2M ZnCl 2 (1CV) with Zn 2 + It was then flushed with distilled water (1.5 CV) and equilibrated with buffer A (2 CV).
[0156] Load the harvest prepared as described above onto an equilibrated IMAC column at a flow rate of 50-150 cm / h with buffer A (5 CV), B - 20...
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