Method for separating specific metal-binding proteins by using protein denaturation

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 ...

AI Technical Summary

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

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