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A method of isolating native sequence nerve growth factor from a mixture

A nerve growth factor and growth factor technology, applied in the field of biotechnology purification, can solve problems such as difficult to remove charge isomers, difficult to remove hydrolyzed misprocessed variants, etc.

Active Publication Date: 2020-10-16
山东衍渡生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the existing β-NGF purification technology solutions are: it is difficult to remove misprocessed variants of hydrolysis, and there are NGF with truncated amino acid sequences in the product; it is difficult to remove various charge isomers (such as carbamylation, oxidation, isoaspartic acid) amino acid, deamidation, etc.)
[0008] But for the natural protein β-NGF without label, how to better separate and purify it is a problem that needs to be solved at present

Method used

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  • A method of isolating native sequence nerve growth factor from a mixture
  • A method of isolating native sequence nerve growth factor from a mixture
  • A method of isolating native sequence nerve growth factor from a mixture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Purification of recombinant human nerve growth factor (expressed in CHO cells) by copper ion chelation chromatography

[0048]Chromatographic column: 1 mL of metal chelating chromatography resin Chelating Sepharose FF (GE Healthcare) was loaded into a GraviTrap PD-10 gravity column (GE Healthcare). Rinse the resin with 5 column volumes (CV) of distilled water, add 0.5 column volume (CV) of 0.1M copper sulfate solution, and Cu 2+ Ions are chelated into the resin.

[0049] Flow rate: 1-1.25mL / min.

[0050] Equilibration buffer: pH 7.5, 50 mM PBS containing 100 mM NaCl.

[0051] Elution buffer: pH8.5, containing 1.5M NH 4 Cl's Tris-HCl Buffer.

[0052] Take 5CV of equilibration buffer and add it to the resin to equilibrate the resin. Take the recombinant human β-NGF culture expressed by CHO cells (see patent application document CN201310016022.2, use the method of Example 2 to transfect the expression vector pCMV-β-NGF-IREs-dhfr prepared by the method of Ex...

Embodiment 2

[0055] Example 2: Purification of recombinant human nerve growth factor (expressed in CHO cells) by zinc ion chelation chromatography

[0056] Chromatographic column: HiTrap Chelating HP 1mL (GE Healthcare), 4 prepacked columns connected in series to make the column height 10cm.

[0057] Equilibration buffer: pH 7.5, 50 mM PBS containing 100 mM NaCl.

[0058] Elution buffer: pH8.5, containing 1.5M NH 4 Cl's Tris-HCl Buffer.

[0059] Flow rate 200cm / h (1.25mL / min).

[0060] Insert the column into Purifier 100 (GE Healthcare), use distilled water to wash the column for 5CV and then wash 1CV with 0.1M zinc sulfate solution to make Zn 2+ Ions are chelated into the resin.

[0061] Equilibrium buffer equilibrates the chromatographic column at 5CV, takes 15 mL of the recombinant human β-NGF supernatant (same as the sample preparation in Example 1) expressed by CHO cells, adjusts the pH to 7.5, and loads the sample into the chromatographic column. Wash 5CV with equilibration bu...

Embodiment 3

[0063] Embodiment 3: Purification of mouse nerve growth factor by nickel ion chelation chromatography

[0064] Sample preparation: Take an appropriate amount of rat submandibular gland, wash and homogenize with a high-pressure homogenizer to remove surface oil. The homogenate was repeatedly frozen and thawed three times, and then the supernatant was collected by centrifugation. Adjust the pH of the supernatant to 4.0 for acid hydrolysis, let it stand at 4°C for 10 minutes, and adjust the pH to 7.5 to obtain the supernatant of the acid hydrolysis of the rat submandibular gland.

[0065] Chromatographic column: HisTrap FF Crude 1mL.

[0066] Equilibration buffer: pH 7.5, 50 mM PBS containing 100 mM NaCl.

[0067] Elution buffer: pH 7.5, 50 mM PBS containing 100 mM NaCl, 150 mM imidazole.

[0068] Use a syringe to draw 5CV of equilibration buffer and inject it into the prepacked column to equilibrate the resin. Aspirate the above-mentioned 20mL rat submandibular gland acid hy...

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Abstract

The invention discloses a method for separating a natural sequence nerve growth factor from a mixture. A solution containing a human or mouse nerve growth factor is separated and purified through metal ion chelating chromatography to obtain the natural sequence nerve growth factor; the metal ion chelating chromatography resin comprises a supporting medium, a chelating ligand and metal ions; the metal ions are chelated on the chelating ligand; the elution condition is to change the concentration gradient of protein and metal ion coordinate bound competitive materials. Strong cation exchange chromatography can be conducted before metal ion chelating chromatography; after metal ion chelating chromatography, efficient strong cation exchange chromatography can be conducted. According to the method, the nerve growth factor with natural sequence and without any protein tags can be purified and separated rapidly.

Description

technical field [0001] The invention relates to the field of biotechnology purification, in particular to a method for isolating natural sequence nerve growth factor from a mixture. Background technique [0002] Nerve growth factor (NGF) is the earliest discovered nervous system trophic factor. It has a wide range of functions and plays an important role in the growth, development, differentiation and regeneration of central and peripheral nerve cells. The β subunit is the active region of NGF. The so-called β-NGF is a mature peptide of 118 amino acids. In the human body, the expression of β-NGF has the following processes: 1. The coding sequence of β-NGF (723bp) is translated into prepro-NGF (241aa), including signal peptide (1-18aa), guide peptide (19-121aa), mature Peptide (122-241aa); 2. On the endoplasmic reticulum, the signal peptide is hydrolyzed to form pro-NGF, including guide peptide and mature peptide. There are 4 amino acid residues (118-121aa) Arg-Ser-Lys-Arg ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/48C07K1/18
CPCC07K14/48
Inventor 张文宇黄奋飞陈振雄马大壮章永垒陈星
Owner 山东衍渡生物科技有限公司
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