CN115304667A

CN115304667A - Method for crude extraction of secretory recombinant human growth hormone

Info

Publication number: CN115304667A
Application number: CN202210911310.3A
Authority: CN
Inventors: 孙晓峰
Original assignee: Changchun Saien Biotechnology Co ltd
Current assignee: Changchun Saien Biotechnology Co ltd
Priority date: 2022-07-29
Filing date: 2022-07-29
Publication date: 2022-11-08

Abstract

The invention provides a method for crude extraction of secretory recombinant human growth hormone, which adopts escherichia coli engineering bacteria to express the recombinant human growth hormone, and target protein is secreted in periplasm, and comprises the following steps: step 1: collecting thalli; step 2: repeated freeze thawing; and step 3: the buffer solution is subjected to hypotonic lysis extraction, and a mild repeated freezing and thawing technology is adopted, so that the cell wall of the engineering bacteria is damaged, and the cell membrane is not damaged, thereby releasing the recombinant human growth hormone target protein from the periplasm, reducing the damage to the whole engineering bacteria caused by a violent cell disruption method, and further reducing the influence on the purity and the biological activity of the target protein caused by the release of nucleic acid and other foreign proteins.

Description

Method for crude extraction of secretory recombinant human growth hormone

Technical Field

The invention relates to the technical field of human growth hormone extraction, in particular to a method for crude extraction of secretory recombinant human growth hormone.

Background

Human growth hormone (hGH) is a protein hormone secreted by anterior pituitary, has a molecular weight of 22KDa and 191 amino acids, has two disulfide bonds, is free of glycosylation modification, and is the most important hormone for promoting growth after birth of human beings. Is mainly used for treating or relieving the children short stature, tissue repair, burn and scald, adult Growth Hormone Deficiency (GHD) and the like caused by the insufficient or deficient secretion of the Growth Hormone (GH). rhGH is an unstable macromolecular protein and is easy to change by oxidation, deamidation, polymerization and the like. The biological activity of growth hormone is reduced after oxidation and deamidation reaction, and the existence of oxidation and deamidation components is considered as an important expression of quality reduction. In addition, growth hormone is prone to form dimers and multimers under severe shock, high temperature, etc., thereby reducing or losing biological activity.

At present, common genetic engineering receptor cells comprise microorganisms such as escherichia coli, bacillus subtilis, saccharomycetes and the like, animal and plant cells and the like. The crude extraction method of the target product mainly comprises the following steps: physical and chemical methods, and physical crushing method includes high pressure homogenizing, bead milling, ultrasonic crushing, repeated freeze thawing, etc. The chemical method comprises an enzymatic method and a chemical reagent method.

The repeated freezing and thawing method is to cool the cell to be disrupted to-15 deg.c to-80 deg.c, to thaw at room temperature, and to swell and disrupt the cell due to the formation of ice grains inside the cell and the increased salt concentration in the residual cytosol.

The engineering bacteria of Escherichia coli belong to gram-negative bacteria, and the cell wall is fragile. Deep freeze thawing easily breaks cell membranes, releases cell contents, particularly swells nucleic acid and some proteolytic enzymes, and has obvious influence on later purification.

Disclosure of Invention

The invention provides a method for crude extraction of secretory recombinant human growth hormone, aiming at the problems in the prior art. The technical scheme of the invention is as follows:

a method for crude extraction of secretory recombinant human growth hormone is characterized in that the recombinant human growth hormone is expressed by adopting engineering bacteria of escherichia coli, and target protein is secreted in periplasm, and the method comprises the following steps:

step 1: collecting thalli, and collecting the engineering thalli of the escherichia coli after fermentation;

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-20-60 ℃, starting timing when the cell temperature is stable at the temperature, and after freezing for 24 hours, placing the cells in a room temperature environment for completely thawing for 1 freezing and thawing; performing the above circulation process for 2 times of freeze thawing, and performing the above circulation process for 3 times of freeze thawing for 1-3 times;

and 3, step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, and performing lysis on the thalli subjected to freeze thawing in the step (2), wherein the method comprises the following steps:

(2) According to the following steps of 1: adding TE buffer solution at a ratio of 8 (w/v) at 4-8 deg.C under stirring speed of 100rpm for 90 min

(2) Centrifuging at 6000rpm for 15min with horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Preferably, in step 2, the collected thallus is placed in an environment with a temperature of-60 ℃, and when the temperature of the thallus is stabilized at-60 ℃, timing is started, and freeze thawing is carried out for 2 times.

Preferably, the TE buffer is composed of 10mmol/L TRIS and 1mmol/L EDTA, pH7.5.

Compared with the prior art, the invention can obtain the following technical effects:

the invention provides a crude extraction method of secretory recombinant human growth hormone, which adopts escherichia coli engineering bacteria to express the recombinant human growth hormone, target protein is secreted in periplasm, and mild repeated freezing and thawing technology is adopted to ensure that the cell wall of the engineering bacteria is destroyed and the cell membrane is not damaged, thereby releasing the recombinant human growth hormone target protein from the periplasm, reducing the damage to the whole engineering bacteria caused by violent cell disruption method, and reducing the influence on the purity and the biological activity of the target protein caused by the release of nucleic acid and other hybrid proteins.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a graph of data relating to freeze-thaw gross extraction of recombinant human growth hormone in various embodiments of the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1:

a method for crude extraction of secretory recombinant human growth hormone comprises the following steps:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-20 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, placing the cells in a room temperature environment for completely thawing, and freezing and thawing for 1 time;

and step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the following steps of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v) at a temperature of 4-8 ℃ at a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Example 2:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-20 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing to be the freeze thawing of the 2 nd time; freezing and thawing for 2 times;

and step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the following steps of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v), at a temperature of 4-8 ℃ and a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Example 3:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-20 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing, namely freezing and thawing for the 1 st time; performing the above circulation process for the 2 nd freeze thawing, and performing the above circulation process for the 3 rd freeze thawing for 3 times;

and step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the proportion of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v) at a temperature of 4-8 ℃ at a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Example 4:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-40 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, placing the cells in a room temperature environment for completely thawing, and freezing and thawing for 1 time;

Example 5:

and 2, step: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-40 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and placing the cells in a room temperature environment for completely thawing to be the freeze thawing of the 2 nd time; freezing and thawing for 2 times;

Example 6:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-40 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing, namely freezing and thawing for the 1 st time; performing the above circulation process for the second freeze thawing, for the third freeze thawing, for 3 times;

and 3, step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the proportion of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v) at a temperature of 4-8 ℃ at a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Example 7:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-60 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing for 1 time;

Example 8:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-60 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing to be the freeze thawing of the 2 nd time; freeze thawing for 2 times;

Example 9:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-60 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing, namely freezing and thawing for the 1 st time; performing the above circulation process for the 2 nd freeze thawing, and performing the above circulation process for the 3 rd freeze thawing for 3 times;

Example 10:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-80 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, placing the cells in a room temperature environment for completely thawing, and freezing and thawing for 1 time;

Example 11:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-80 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing to be the freeze thawing of the 2 nd time; freeze thawing for 2 times;

and 3, step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the following steps of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v) at a temperature of 4-8 ℃ at a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with a horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

Example 12:

step 2: repeatedly freezing and thawing, namely placing the cells collected in the step 1 in a constant temperature environment of-80 ℃, starting timing when the cell temperature is stable at the temperature, freezing for 24 hours, and then placing the cells in a room temperature environment for completely thawing, namely freezing and thawing for the 1 st time; performing the above circulation process for the 2 nd freeze thawing, and performing the above circulation process for the 3 rd freeze thawing for 3 times;

and step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, wherein the step of lysing the thalli after freeze thawing in the step 2 comprises the following steps: (1) according to the following steps of 1: TE buffer (10 mmol/L TRIS,1mmol/L EDTA, pH 7.5) was added at a ratio of 8 (w/v) at a temperature of 4-8 ℃ at a stirring speed of 100rpm for 90 minutes. (2) Centrifuging at 6000rpm for 15min with a horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

In the above examples, the TE buffer components were 10mmol/L TRIS and 1mmol/L EDTA, pH7.5, and experimental analyses were performed on examples 1 to 12

The analysis method 1 comprises the steps of repeatedly freezing and thawing, performing hypotonic lysis, performing low-permeability extraction, and counting viable bacteria. Viable bacteria count was performed by referring to the plate colony method described in "handbook of microbiology experiments". The freeze-thaw breakage rate of the engineering bacteria detected by the analysis method 1 is shown in table 1:

TABLE 1 Freeze-thaw disruption table for engineering bacteria

And (2) detecting by using an ultraviolet spectrophotometry, taking the low-permeability extract or the supernatant OD260 and OD280 prepared in the step (3), and if OD260/OD280 is more than 1, indicating that the content of nucleic acid is high, the release amount of cell contents is increased, and freeze-thaw overload is caused. Protein content was determined by lowry method. Formula C = (1.45 × OD280-0.74 × OD 260) × 1.23 × dilution factor

Analysis method 3: detecting the purity of the target protein by using high performance liquid chromatography. Sepax C4 chromatographic column, 4.6X 250mm,5um, detecting the content of related protein.

The protein content and purity measured by

analytical methods

2 and 3 are shown in table 2:

TABLE 2 destination protein content and purity table

Result analysis shows that the judgment standard is 1, the higher the thallus breakage rate is, the fewer the viable bacteria are, the better the index is, and the general breakage rate is more than 90 percent; 2. ensure that the ratio of OD280 to OD260 is greater than 1; 3. the target protein content and purity are at relatively high stages. Through the analysis of the relevant data in tables 1 and 2, example 8 is the best example, and example 8 is to freeze-thaw the collected materials in step 1 for 2 times under the constant temperature environment of-60 ℃.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of additional like elements in a commodity or system comprising the element.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for crude extraction of secretory recombinant human growth hormone is characterized in that the recombinant human growth hormone is expressed by adopting engineering bacteria of escherichia coli, and target protein is secreted in periplasm, and the method comprises the following steps:

and step 3: and (3) performing hypotonic lysis extraction by using a buffer solution, and performing lysis on the thalli subjected to freeze thawing in the step (2), wherein the method comprises the following steps:

(1) According to the following steps of 1: adding TE buffer solution at a ratio of 8 (w/v) at 4-8 deg.C under stirring speed of 100rpm for 90 min

(2) Centrifuging at 6000rpm for 15min with a horizontal centrifuge to obtain supernatant as low-permeability extractive solution.

2. The method for the crude extraction of the secretion type recombinant human growth hormone as claimed in claim 1, wherein the collected thallus is placed in a temperature environment of-60 ℃ in the step 2, and when the temperature of the thallus is stabilized at-60 ℃, the timing is started and the freeze thawing is performed for 2 times.

3. The method of claim 2, wherein TE buffer comprises 10mmol/L TRIS and 1mmol/L EDTA, pH7.5.