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Method for modifying, separating and purifying protein and method for regulating enzyme activity of sulfhydryl protease

A technology for the separation and purification of thiol proteases, which is applied to the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., which can solve the problems of troublesome operation and high cost

Inactive Publication Date: 2019-02-26
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods need to add reagents all the time to maintain the sulfhydryl group of protease from oxidation damage, which is costly and troublesome to operate

Method used

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  • Method for modifying, separating and purifying protein and method for regulating enzyme activity of sulfhydryl protease
  • Method for modifying, separating and purifying protein and method for regulating enzyme activity of sulfhydryl protease
  • Method for modifying, separating and purifying protein and method for regulating enzyme activity of sulfhydryl protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Modification and separation and purification of protein

[0038] 1. Protein-polymer preparation (protein modification)

[0039] 1 Reduced sulfhydryl groups on the surface of TCEP protein

[0040] 1) Dissolve 80mg of BSA and 11mg of TCEP in 10mL of acetic acid buffer at a molar ratio of 1:1, respectively, and connect to a nitrogen deoxygenation device for deoxygenation for 7 minutes; obtain TCEP solution and protein solution respectively;

[0041] 2) Slowly add TCEP to the protein solution and react with magnetic stirring for 7 minutes under the condition of nitrogen deoxygenation. Phosphate buffer was used twice, and a nitrogen deoxygenation device was connected throughout the dialysis process. After the dialysis, the liquid in the dialysis bag was collected, that is, dialysate A.

[0042] 2 Initiator for protein surface sulfhydryl modification

[0043] 1) Take 10mL dialysate A and add it to a weighing bottle of appropriate size, add 2mL Buffer 8.2 and adju...

Embodiment 2

[0056] Example 2 Enzyme activity regulation of thiol protease

[0057] 1. Enzyme activity inhibition of thiol protease

[0058] 1 Reduced sulfhydryl groups on the surface of TCEP protein

[0059] 1) Dissolve 80mg of thiol protease protein (Bromelain and Papain were used as examples in this example) and 11mg of TCEP in 10mL of acetic acid buffer at a molar ratio of 1:1, respectively, and connect to a nitrogen deoxygenation device to remove Oxygen for 7min; obtain protease solution and TCEP solution respectively;

[0060] 2) Slowly add the TCEP solution to the protein solution dropwise, and react with magnetic stirring for 7 minutes under the condition of nitrogen deoxygenation. After the reaction, transfer the mixed solution into a dialysis bag, put the dialysis bag into the buffer solution and dialyze three times, and use acetate buffer solution for the first time. Phosphate buffer was used for the last two times, and the nitrogen deoxygenation device was connected to the wh...

Embodiment 3

[0074] Papain and bromelain were used to protect the activity of thiol proteases by polymers, and four different treatment methods were used to compare the enzyme activities.

[0075] according to image 3 Determine the concentration of Papain, Papain-NIPAM polymer, Bromelain and Bromelain-NIPAM polymer with the standard curve of protein concentration, and adjust the concentration to 0.1mg / mL to measure the enzyme activity, because the absorbance value of casein at this concentration is measured in the ultraviolet Within the appropriate range that can be measured by a spectrophotometer.

[0076] 1) The four processing methods are as follows:

[0077]Papain, Papain-NIPAM polymers and bromelain, Bromelain-NIPAM polymers are treated in the same way, and papain is taken as an example below.

[0078] a) pH2.0 treatment

[0079] Considering factors such as sample size, experimental error, and convenient pH adjustment, each take a certain amount of Papain and Papain-NIPAM polymer ...

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Abstract

The invention discloses a method for modifying, separating and purifying protein and a method for regulating enzyme activity of sulfhydryl protease. A sulfhydryl group of the protein is grafted with apolymer macromolecule; by using the critical temperature characteristic of a polymer NIPAM at 32 DEG C, when the temperature exceeds 32 DEG C, a protein-polymer is separated out from the solution, and then the protein-polymer is centrifugally precipitated; and the pure protein-polymer is treated with DTT or another thiol reducing agent, and the macromolecule is cut off to obtain the an original protein, thus achieving the purpose of purifying a target protein. The sulfhydryl group exposed on the surface of the sulfhydryl protease is closely related to the enzyme activity of the sulfhydryl protease, and the enzyme activity will also be lost after the sulfhydryl group on the surface is destroyed. When the protein is sulfhydryl protease, the method can realize regulation of the enzyme activity.

Description

technical field [0001] The invention discloses a method for modifying, separating and purifying proteins and regulating the enzyme activity of sulfhydryl protease. Background technique [0002] At present, there are many methods for protein separation and purification: (1) Separation and purification according to different molecular sizes, which can be divided into dialysis, ultrafiltration, centrifugation and gel filtration; (2) Separation and purification according to different solubility, commonly used methods are: Isoelectric point precipitation and pH value adjustment, protein salt dissolution and salting out, organic solvent method, two-phase extraction method, reverse micellar extraction method, etc.; (3) The method of separating proteins according to the different charges of proteins, that is, the acid-base properties , There are two types of electrophoresis and ion exchange chromatography. The above separation methods are cumbersome or incomplete, and some require ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/47C07K1/107C07K1/30C07K1/34C12N9/50
CPCC07K14/4732C07K14/765C12N9/63C12Y304/22002C12Y304/22004
Inventor 吴元子蔡振巫水根熊文丽马山云
Owner FUZHOU UNIV
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