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A method for separating specific metal-binding proteins by protein denaturation

A metal-binding protein and protein denaturation technology, applied in the field of protein chemistry, can solve the problems of metal-binding proteins that cannot be separated, difficult to separate metal-binding proteins, and incomplete separation, and achieve stable complexes, avoid aggregation and precipitation, and simple procedures Effect

Inactive Publication Date: 2018-05-29
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the environment polluted by heavy metals, the metal binding sites of proteins in plant cells may be occupied by metal ions that enter the cells in advance, so that the proteins have no redundant metal binding sites to bind to the fixed metals on the affinity chromatography column, resulting in metal Bound protein cannot be isolated or is incompletely isolated
More importantly, the metal-binding sites of most proteins exist inside the molecule, so in the natural state, it is difficult for us to isolate specific metal-binding proteins by IMAC method, but none of the previous research literature on metal-binding proteins take this into consideration

Method used

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  • A method for separating specific metal-binding proteins by protein denaturation
  • A method for separating specific metal-binding proteins by protein denaturation
  • A method for separating specific metal-binding proteins by protein denaturation

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Embodiment 1

[0034] 1. Extraction of plant protein

[0035] Use 100 μmol L -1 The germinated rice seed embryos treated with copper sulfate were taken 5 days after the copper treatment, and the fresh rice seed embryos were fully ground with liquid nitrogen, then added 4 times the volume of protein extract solution, mixed well, and extracted at 4 °C for 30 min. Centrifuge at 15,000×g for 30 min at 4°C, and collect the supernatant. The denatured protein was quantified by a modified Bradford method (Ramagli, 1999) with chicken ovalbumin as the standard protein. The composition of the protein extract is: every liter of protein solution contains 50mmol / L sodium phosphate, pH 7.8, 300mmol / L sodium chloride, 0.1-1% Triton X-100 by mass percentage, 8mol / L urea, The remainder is water.

[0036] 2. Cu-NTA agarose affinity chromatography process

[0037] (1) Packing: Add 2 ml bed volume of Ni-NTA agarose (GE Healthcare) into the glass tube along the tube wall, and push the piston to an appropriate...

Embodiment 2

[0046] A method for separating a specific metal-binding protein by protein denaturation, comprising the following steps:

[0047] (1) Take biological tissues or cells and grind them fully with liquid nitrogen, add protein extract, mix well, and centrifuge at low temperature to obtain denatured protein solution; The solid-to-liquid ratio of the extract is: 1:4; the composition of the protein extract is: every liter of protein solution contains 50 mmol / L sodium phosphate, pH7.8, 300 mmol / L sodium chloride, and the mass percentage is 0.1 -1% Triton X-100, 8mol / L urea, the balance is water;

[0048] (2) Ni in the purchased Ni-NTA agarose (GE Healthcare) 2+ Replaced with Cu 2+ Load it into a chromatography column; inject the denatured protein solution obtained in the above step (1) into Cu-NTA agarose at a flow rate of 0.25ml / min, and incubate at 37°C for 15-40min to fully bind the denatured protein; Then, under low temperature conditions, slowly inject eluent 1 at a flow rate o...

Embodiment 3

[0057] A method for separating a specific metal-binding protein by protein denaturation, comprising the following steps:

[0058] (1) Take biological tissues or cells and grind them fully with liquid nitrogen, add protein extract, mix well, and centrifuge at low temperature to obtain denatured protein solution; The solid-to-liquid ratio of the extract is: 1:4; the composition of the protein extract is: every liter of protein solution contains 50 mmol / L sodium phosphate, pH7.8, 300 mmol / L sodium chloride, and the mass percentage is 0.2 % Triton X-100, 8mol / L urea, the balance is water;

[0059] (2) Replace the Ni in the purchased Ni-NTA agarose (GE Healthcare) with Cu and load it into the chromatographic column; inject the denatured protein solution obtained in the above step (1) into the Cu- NTA agarose, incubate at 37°C for 15-40min to fully bind the denatured protein; then slowly inject eluent 1 at a flow rate of 0.25ml / min at low temperature to refold the protein and remov...

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Abstract

The invention discloses a method for separating specific metal-binding proteins by using protein denaturation, and relates to the technical field of protein chemistry. The method comprises the following steps: firstly, taking a biological tissue or cell, after fully grinding with liquid nitrogen, adding protein extracting liquid, and after uniformly mixing, carrying out low-temperature centrifugation, so as to obtain a denatured protein solution; replacing Ni in Ni-NTA agarose with Cu, and putting into a chromatographic column; incubating the denatured protein solution with Cu-NTA agarose, so as to ensure that the denatured proteins are fully combined with the Cu-NTA agarose; then under the low-temperature condition, injecting eluant 1, so as to renature the proteins and elute non-specific copper-binding proteins; injecting eluant 2, so as to separate specific copper-binding proteins. According to the method, the copper-binding proteins under the denaturing condition are separated by using agarose-NTA, so that the obtaining efficiency of the copper-binding proteins is greatly improved; a technology suitable for separating the specific metal-binding proteins from the biological tissue or cell under the excess heavy metal treatment condition is established.

Description

technical field [0001] The invention relates to the technical field of protein chemistry, in particular to a method for separating a specific metal-binding protein by protein denaturation. Background technique [0002] The National Pollution Survey Bulletin shows that the total rate of soil pollution in the country is 16.1%, of which heavy metal pollution accounts for more than 80%. Most of the heavy metals in the soil are easy to accumulate in plants and endanger human health through the food chain. . On the other hand, because the cysteine, methionine, and histidine residues of proteins have high affinity for divalent metal ions, excessive heavy metals in cells may interfere with the binding of essential metal elements to proteins, resulting in Protein denaturation or enzyme inactivation interferes with normal metabolic processes. [0003] Immobilized metal affinity chromatography (IMAC) coupled with mass spectrometry is an important technique for the identification of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C07K14/415
Inventor 张红晓张羽宋玉峰王发园王世华
Owner HENAN UNIV OF SCI & TECH
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