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Small molecular compound N-alkyl acyl cystamine for simulating function of folding enzyme, a preparation method thereof and method for assisting protein oxidizing and refolding

A technology of small molecule compound, alkyl cysteine, which is applied in the field of protein renaturation in biotechnology, can solve the problem of not being able to effectively simulate specific interactions, not having the specificity of protein oxidation, and the catalytic effect of small molecule mimics It can save the time and cost of renaturation, reduce the cost of renaturation, and assist the oxidative renaturation of proteins.

Active Publication Date: 2010-12-22
南通药享科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalytic effect of the obtained small molecule mimics is far lower than that of the actual foldase, and a high concentration is required to achieve the effect of assisted refolding
In addition, these small molecules are not able to effectively mimic the specific interactions of PDI and DsbA on denatured proteins
Since these small molecule mimics and the most commonly used redox agents, oxidized glutathione (GSSG) and reduced glutathione (GSH) are not specific for protein oxidation, they are easily destroyed by the strong reducing agent dithiothreo Therefore, when using these small molecules to assist protein refolding, it is necessary to remove the residual reducing agent dithiothreitol in the reduced denatured protein through gel filtration chromatography or dialysis, so that the refolding Complicated sexual process
[0004] In order to overcome the deficiencies of commonly used oxidant-assisted renaturation, the present invention proposes a new type of small molecule compound that mimics the function of foldase and its method of assisting protein renaturation. The amount of oxidant is greatly reduced, and the operation is simple, without the need to separate the denaturant operation steps, saving refolding time and cost

Method used

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  • Small molecular compound N-alkyl acyl cystamine for simulating function of folding enzyme, a preparation method thereof and method for assisting protein oxidizing and refolding
  • Small molecular compound N-alkyl acyl cystamine for simulating function of folding enzyme, a preparation method thereof and method for assisting protein oxidizing and refolding
  • Small molecular compound N-alkyl acyl cystamine for simulating function of folding enzyme, a preparation method thereof and method for assisting protein oxidizing and refolding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Synthesis of N-octylcystamine and its application in renaturation of assisted reduction denatured lysozyme

[0031]Weigh 0.225g of cystamine dihydrochloride and dissolve it in 10mL of deionized water to obtain a 0.1mol / L cystamine solution, then drop in 3.18μL of n-octanoic acid to make the concentration 0.002mol / L, mix well, and add 2mol / L NaOH The pH of the solution was adjusted to 6.3, and then 0.384 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was added thereto to make the final concentration 0.2 mol / L, and mixed quickly and uniformly. The reaction system was placed in a constant temperature water bath shaker at 25°C, and reacted for 4 hours to obtain a crude product. The crude product was filtered through a 0.45 μm water-based microporous membrane, and then separated by high-performance liquid chromatography on a C18 reverse-phase column. The mobile phase A was an aqueous phase containing 0.1% trifluoroacetic acid (TFA), and B was acet...

Embodiment 2

[0033] Embodiment 2: the renaturation of N-octylcystamine assisted reduction denatured lysozyme synthesized in embodiment 1

[0034] The N-octylcystamine prepared in Example 1 was used to assist the renaturation of the reduced denatured lysozyme. 14.3mg of lysozyme was dissolved in 1.0mL containing 8mol / L urea, 100mmol / L DTT, 100mmol / L tris-hydrochloride (Tris-HCl) and 1mmol / L EDTA, in the denaturing buffer solution of pH 8.5, Mix well and reduce and denature at 40°C for 3 hours; after the denaturation is completed, the residual DTT content is 84mmol / L as determined by C18 reverse phase chromatography; the obtained denatured lysozyme is diluted 5 times with DTT-free denaturing buffer Denatured protein samples. The refolding buffer was composed of 100mmol / L Tris-HCl, 2mol / L urea, 1mmol / L EDTA and 0.2mmol / L N-octylcystamine or cystamine, and the pH of the solution was 8.5. The denatured protein sample was diluted 40 times with refolding solution, the concentration of lysozyme ...

Embodiment 3

[0035] Example 3: Synthesis of N-heptylcystamine and its renaturation for auxiliary reduction of denatured ribonuclease A

[0036] Weigh 2.25g of cystamine dihydrochloride and dissolve it with 10mL of deionized water to obtain a 1mol / L cystamine solution, then add 28.6μL of n-heptanoic acid dropwise to make the concentration 0.02mol / L, mix well, and dissolve with 5mol / L NaOH The pH of the solution was adjusted to 6.5, and then 0.192 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was added thereto to make the final concentration 0.1 mol / L, and mixed quickly and uniformly. The reaction system was placed in a constant temperature water bath shaker at 15°C, and reacted for 6 hours to obtain a crude product. The crude product was filtered through a 0.45 μm water-based microporous membrane, and then separated by high-performance liquid chromatography on a C18 reverse-phase column. The mobile phase A was an aqueous phase containing 0.1% trifluoroacetic acid (TFA), an...

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Abstract

The invention relates to a small molecular compound N-alkyl acyl cystamine for simulating the function of folding enzyme, a preparation method thereof and a method for assisting protein oxidizing and refolding. The small molecule is used as an oxidizing agent, so the small molecular compound can promote the formation of protein disulfide even in the strongly reducing environment, and an operatingstep of removing a strong reducer DTT from reduced and denatured protein solution is saved; therefore, the refolding time and cost are saved. The small molecular compound N-alkyl acyl cystamine for simulating the function of the folding enzyme can greatly improve the protein oxidizing and refolding speed which is 7 to 10 times that of oxidized L-glutathione for assisting refolding under the same conditions; the N-alkyl acyl cystamine can effectively assist protein oxidizing and refolding under lower concentration, and the consumption of the oxidizing agent can be greatly reduced; and the adopted N-alkyl acyl cystamine can be coupled with strong reducer carried by the denatured and reduced protein to form an redox couple, so the reducer is not needed to be added into refolding solution, the operation is simple and the refolding cost is reduced.

Description

technical field [0001] The invention relates to a small molecular compound that simulates the function of foldase, a synthesis method thereof, and a method for using the same in assisting protein oxidative renaturation, and belongs to the technical field of protein renaturation in biotechnology. Background technique [0002] Protein folding renaturation is not only related to some human diseases, but also a bottleneck in the production of valuable pharmaceutical proteins using gene recombination technology. Therefore, protein folding and renaturation technology is a hot research topic at present. Many important pharmaceutical proteins contain disulfide bonds, such as human growth hormone, human interferon, tumor necrosis factor, insulin, interleukin, tissue plasminogen activator, etc. The folding renaturation of these disulfide bond-containing proteins is a great challenge in protein renaturation. The folding process of proteins containing disulfide bonds is called oxidati...

Claims

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Application Information

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IPC IPC(8): C07C323/41C07C319/22C07K1/00
Inventor 孙彦王国珍史清洪董晓燕
Owner 南通药享科技有限公司
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