CN110669673A

CN110669673A - Method for extracting saccharomyces cerevisiae EST3 telomerase activation factor

Info

Publication number: CN110669673A
Application number: CN201810707985.XA
Authority: CN
Inventors: 王虎
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2020-01-10

Abstract

The invention discloses a method for extracting a saccharomyces cerevisiae EST3 telomerase activation factor, which comprises the following steps: dissolving Saccharomyces cerevisiae in physiological saline to obtain suspension, adding yeast composite wall-breaking enzyme and dissolving promoter, and heating at 45-55 deg.C for 24 hr. Injecting the self-dissolving wall-broken brewing yeast solution into an intelligent high-speed ball mill, selecting diamond as collision impact ball beating, performing high-speed impact crushing on the yeast in the yellow wine yeast suspension by using the diamond ball through a high-speed rotary ball mill to realize 95% wall breaking and content release of the yeast, filtering the obtained suspension, adding multifunctional ribonucleic acid protease, and incubating at incubation temperature; EST3 telomerase activating factor was purified and electrophoretically extracted. The preparation method of the invention fully releases EST3 telomerase activation factor components, and extracts EST3 telomerase activation factor with the purity of 98 percent through further nano purification and electrophoresis.

Description

Method for extracting saccharomyces cerevisiae EST3 telomerase activation factor

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for breaking cell wall of saccharomyces cerevisiae and extracting and purifying a novel telomerase activation factor.

Background

Telomeres, which are called "life clocks", have been hot spots in the fields of aging and cancer (since 1929, nine scientists have achieved the Nobel prize in their research results), and their length is closely related to their longevity. With the increase of the cell division times, telomeres are shortened, and telomerase is a key enzyme for maintaining and supplementing the length of the telomeres. And Saccharomyces cerevisiae contains at least five telomerase activating factors. The method comprises the following steps: EST1p, EST2p, EST3p, TLC1, CDC 13.

Telomeres (Telomere) are specific structures of the chromosome ends of eukaryotic cells. Human telomeres are composed of 6 base repeats (TTAGGG) and binding proteins. Telomeres have important biological functions, can stabilize the functions of dyeing telomerase chromosomes, prevent the degradation and end fusion of chromosome DNA, protect the DNA of chromosome structural genes and regulate the growth of normal cells. Telomeres (Telomere) are ribonucleic acid-protein complexes consisting of RNA and protein. The RNA component is used as a template, the protein component has catalytic activity, and the 5' end of the telomere is used as a primer to synthesize a telomere repetitive sequence. The invention uses innovative bioengineering technology to extract the telomerase activation initiation factor with full ecology and full gene activity as a core product, changes and prolongs the life clock, innovating medical tests, drug research and treatment, realizes the conversion from a fuzzy life cycle concept to an accurate life clock, and improves the accuracy and precision of health indexes and the efficiency of life health care, thereby having scientific significance for greatly improving the population quality and the health level.

The human life ark program started in the beginning of the 21 st century made a breakthrough progress in the study of the cellular aging process, which makes it appear that the cancer-curing eosin is seen. In fact, this enzyme is also of concern in that it has been found to be present in 85% of tumors, possibly culminating in the unregulated proliferation of cancer cells. Nevertheless, it is fully possible to develop new technologies that can prevent aging and overcome cancer, as long as the researchers are fully aware of the principle of action. Proc. Jie-Shei and Wudrin-Reiter, professor of the science of cell biology and nervous system, the university of Texas, USA, made an experiment by introducing into collected foreskin cells (the by-product of circumcision) Telomerase a gene which makes the cells produce the enzyme Telomerase (Telomerase). Generally, foreskin cells can divide about 60 times before becoming old. However, in the above experiment, the cells had divided over 300 times without signs of termination and did not show any signs of abnormality. "cells divide and reproduce under the action of telomerase, as in small rabbits injected with stimulants, in Shei. At the same time, researchers of Geron corporation, a partner of Shie and Reit, USA, performed the same experiment using human retinal cells. As a result, these cells also appear to become immature. Researchers have thus seen an attractive hope, but they do not know if this method of controlling the cellular senescence process could ultimately be equally effective in delaying senescence in humans. Thus, to date, no proposal has been made to add telomerase to our daily diet.

Disclosure of Invention

A. Cell wall breaking of saccharomyces cerevisiae: the multi-group compound enzyme and the polypod yeast wall-breaking promotion solvating agent are used, and the mechanical wall-breaking technology is applied to cell wall breaking and content release of saccharomyces cerevisiae. EST3 telomerase activating factor and nucleic acid are both located in yeast cells, and to extract EST3 telomerase activating factor, an effective method must be adopted to break the walls of the yeast cells so as to realize the full release of the EST3 telomerase activating factor and avoid the inactivation of the EST3 telomerase activating factor and the nucleic acid. The protein engineering transformation is carried out on the traditional protease by adopting an enzyme bioengineering technology, so that the novel EST3 telomerase activating factor with better enzymolysis performance is obtained, and the enzyme-assisted mechanical wall breaking extraction is carried out on the saccharomyces cerevisiae by utilizing a controllable enzymolysis technology and an intelligent high-speed mechanical wall breaking technology, so that the optimization of the degradation of the EST3 telomerase activating factor protein is achieved.

B. Extraction and purification of EST 3: mixing the wall-broken cell extract of saccharomyces cerevisiae and normal saline according to the ratio of 1: 3, heating to 45-55 ℃, quickly cooling to below 18 ℃, adjusting the pH value to 2.8 by using hydrochloric acid, and centrifuging to collect supernatant.

C. The membrane separation technology is applied to the compatible efficient separation and purification of EST3 telomerase activating factor. In the process of yeast cell breaking, EST3 telomerase activating factor is released, and some protein and polypeptide molecules in the cell are released simultaneously. According to researches, certain protein and polypeptide molecules can interfere with the separation and purification of EST3 telomerase activating factors, reduce the recovery rate of EST3 telomerase activating factors, degrade EST3 telomerase activating factors and remarkably reduce the yield of EST3 telomerase activating factors. Therefore, how to eliminate the interference of the substances and ensure the recovery rate of the EST3 telomerase activating factor is a key technical problem for preparing the EST3 telomerase activating factor from the saccharomyces cerevisiae. Because the saccharomyces cerevisiae cell-breaking liquid contains impurities such as protein, polypeptide, oligopeptide and the like which interfere the separation and purification of the EST3 telomerase activation factor, the traditional ion exchange resin adsorption method has the defects of low adsorption efficiency, low recovery rate of the EST3 telomerase activation factor and low product purity when separating glutathione. Meanwhile, in order to synchronously extract EST3 telomerase activating factors in saccharomyces cerevisiae, innovative modification must be carried out on the traditional extraction method. Aiming at the problem, the separation and purification method of the EST3 telomerase activation factor is innovated from two aspects, and a compatible efficient separation and purification technology of the EST3 telomerase activation factor is developed. EST3 telomerase activating factor belongs to two completely different substances, and has great difference in properties such as molecular weight, charge and the like.

D. Electrophoresis is highly purified.

The method for extracting EST3 telomerase activating factor by using saccharomyces cerevisiae has high yield, the obtained product has high purity, keeps the bioactivity, realizes the comprehensive utilization of resources, improves the resource utilization level of the wine fermentation industry, reduces the production cost, and is suitable for large-scale industrial production.

Example 1

1) Breaking the wall of the yeast: putting 1000g of active yellow wine saccharomyces cerevisiae into 3000g of physiological saline, adjusting the pH value to 9 with sodium bicarbonate, stirring for 60 minutes, centrifuging and washing for three times to neutrality, dispersing 900g of the obtained precipitated yeast into 900g of physiological saline, adding 10g of lysozyme, 8 g of mannitol enzyme, 15g of sodium dodecyl sulfate and 11g of sodium bicarbonate, heating to 45 ℃, reacting for 6 hours, adding 100g of ethanol, stirring for 60 minutes, putting the colloidal solution into a supercritical reaction kettle, controlling the pressure of the reaction kettle to be 10 MPa, the temperature to be 45 ℃, and the yield to be 46.6%.

2) EST3 telomerase activation factor purification: the silica gel resin capable of specifically adsorbing nucleic acid is used for adsorbing the EST3 telomerase activating factor, so that the high-purity and high-yield extraction of the EST3 telomerase activating factor is realized, and the yield of the EST3 telomerase activating factor reaches 0.5%. The specific adsorption of EST3 telomerase activating factor is realized by adopting silica gel column adsorption (high salt and low pH value), protein impurities are removed, and then the EST3 telomerase activating factor is washed by 95% ethanol column washing and 55 ℃ sterile water, and the purity of the product is more than 80%.

3) Electrophoresis EST3 telomerase activating factor purification, wherein the adding amount of chemical reagent for purifying the telomerase activating factor is 90mM ethylene diamine tetraacetic acid; 80mM Tris. Cl; 20% tween 20; 1.6% triton X-100; 600mM guanidinium salt; 2% SDS); the incubation temperature is 45 to 55 ℃; the incubation time is 24 minutes to 48 hours, and the purity reaches 98.5 percent after electrophoresis.

Example 2

1) Breaking the wall of the yeast: putting 1000g of active yellow wine saccharomyces cerevisiae into 3000g of physiological saline, adjusting the pH value to 9 with sodium bicarbonate, stirring for 60 minutes, centrifuging and washing for three times to neutrality, dispersing 900g of the obtained precipitated yeast into 900g of physiological saline, adding 15g of lysozyme, 12 g of mannitol enzyme, 20g of sodium dodecyl sulfate and 15g of sodium bicarbonate, heating to 45 ℃, reacting for 6 hours, adding 100g of ethanol, stirring for 60 minutes, putting the colloidal solution into a supercritical reaction kettle, controlling the pressure of the reaction kettle to be 10 MPa, the temperature to be 45 ℃, and the yield to be 50.6%.

2) EST3 telomerase activation factor purification: the silica gel resin capable of specifically adsorbing nucleic acid is used for adsorbing the EST3 telomerase activating factor, so that the high-purity and high-yield extraction of the EST3 telomerase activating factor is realized, and the yield of the EST3 telomerase activating factor reaches 0.6%. The specific adsorption of EST3 telomerase activating factor is realized by adopting silica gel column adsorption (high salt and low pH value), protein impurities are removed, and then 96% ethanol is used for washing the EST3 telomerase activating factor by 55 ℃ sterile water, so that the purity of the product is as high as 85%.

3) Electrophoresis EST3 telomerase activating factor purification, adding 80mM ethylene diamine tetraacetic acid into a chemical reagent for purifying the telomerase activating factor; 70mM Tris & Cl; 18% tween 20; 2% triton X-100; 500mM guanidine salt; 2% SDS); the incubation temperature is 45 to 55 ℃; the incubation time is 24 minutes to 48 hours, and the purity reaches 97.5 percent after electrophoresis.

Example 3

1) Breaking the wall of the yeast: putting 1000g of active yellow wine saccharomyces cerevisiae into 3000g of physiological saline, adjusting the pH value to 9 with sodium bicarbonate, stirring for 60 minutes, centrifuging and washing for three times to neutrality, dispersing 900g of the obtained precipitated yeast into 900g of physiological saline, adding 20g of lysozyme, 15g of mannitol enzyme, 25g of sodium dodecyl sulfate and 20g of sodium bicarbonate, heating to 45 ℃, reacting for 6 hours, adding 120g of ethanol, stirring for 60 minutes, putting the colloidal solution into a supercritical reaction kettle, controlling the pressure of the reaction kettle to be 10 MPa, the temperature to be 45 ℃, and the yield to be 45.6%.

2) EST3 telomerase activation factor purification: the silica gel resin capable of specifically adsorbing nucleic acid is used for adsorbing the EST3 telomerase activating factor, so that the high-purity and high-yield extraction of the EST3 telomerase activating factor is realized, and the yield of the EST3 telomerase activating factor reaches 0.48 percent. The specific adsorption of EST3 telomerase activating factor is realized by adopting silica gel column adsorption (high salt and low pH value), protein impurities are removed, and then the EST3 telomerase activating factor is washed by 93 percent ethanol column washing and 55 ℃ sterile water, and the purity of the product is as high as 80 percent.

3) Electrophoresis EST3 telomerase activating factor purification, adding 60mM EDTA as chemical reagent for purifying telomerase activating factor; 60mM Tris. Cl; 15% tween 20; 1% triton X-100; 400mM guanidinium salt; 1.5% SDS); the incubation temperature is 45 to 55 ℃; the incubation time is 24 minutes to 48 hours, and the purity reaches 96.5 percent after electrophoresis.

Drawings

FIG. 1 shows the EST3 purified protein obtained in example 1. Protein detection map shows EST3 telomerase activator purified protein (molecular weight 25.5 KD).

Claims

1. A method for extracting a saccharomyces cerevisiae EST3 telomerase activating factor comprises the following steps: adding appropriate amount of composite wall-breaking enzyme and combined dissolving promoter into active Saccharomyces cerevisiae. Firstly, heating and autolysis, then injecting autolysis wall-broken saccharomyces cerevisiae into an intelligent high-speed spherical grinder, then dissolving multifunctional ribonucleic acid protease into buffer solution, incubating overnight at 45-55 ℃, and then applying membrane separation and electrophoresis technology to efficiently separate and purify EST3 telomerase activation factor compatibility.

2. The method for extracting the saccharomyces cerevisiae EST3 telomerase activating factor as claimed in claim 1, wherein: the composite yeast broken wall complex enzyme comprises 1-2% of lysozyme (enzyme activity 8000 units) and 0.5-15% of mannitol enzyme (enzyme activity 12000 units).

3. The method for extracting the saccharomyces cerevisiae EST3 telomerase activating factor as claimed in claim 1, wherein: the combined yeast cell wall breaking and dissolving promoting agent is a mixture of 1.5-2% of sodium dodecyl sulfate and 0.8-1.2% of sodium bicarbonate, the temperature is raised to 45-55 ℃, and the reaction time is 24-48 hours.

4. The method for extracting the saccharomyces cerevisiae EST3 telomerase activating factor as claimed in claim 1, wherein: the internal environment of the intelligent high-speed spherical polisher is between 8 and 12MPa of vacuum negative pressure, and the diameter of the diamond ball is 0.2 to 1.6 mm.

5. The method for extracting the saccharomyces cerevisiae EST3 telomerase activating factor as claimed in claim 1, wherein: according to the membrane separation technology, the diameter of a membrane pore is 1 micron.

6. The method for extracting the saccharomyces cerevisiae EST3 telomerase activating factor as claimed in claim 1, wherein: the chemical reagent for purifying the telomerase activating factor is added with 90mM ethylene diamine tetraacetic acid; 80mM Tris. Cl; 20% tween 20; 1.6% triton X-100; 600mM guanidinium salt; 2% SDS); the incubation temperature is 45 to 55 ℃; the incubation time is 24 minutes to 48 hours.