CN113684168B

CN113684168B - Culture medium and preparation method of escherichia coli exocytosis recombinant protein

Info

Publication number: CN113684168B
Application number: CN202111026999.3A
Authority: CN
Inventors: 冯建敏; 赵占勇; 郑长龙
Original assignee: Beijing Dacheng Biotechnology Co ltd
Current assignee: Beijing Dacheng Biotechnology Co ltd
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2023-03-17
Anticipated expiration: 2041-09-02
Also published as: CN113684168A

Abstract

The invention discloses a culture medium, wherein each liter of the culture medium comprises 5-15g of triton, 12.5-50g of cane sugar, 5-15g of pyrophosphate and the balance of LB liquid culture medium. The invention also discloses a preparation method of the escherichia coli extracellular secretion recombinant protein, which comprises the following steps: s1, inoculating a seed solution containing an expression strain into an LB liquid culture medium to be cultured until the thallus concentration OD _600nm When the concentration is 1.8-2.2, adding triton, sucrose and IPTG, wherein the concentration of the IPTG is 50 mu mol/L; s2, setting the culture temperature to be 25 ℃, adding pyrophosphate into the culture medium in the step S1, continuing to culture for a preset time, and then stopping fermentation; s3, taking the supernatant and purifying to obtain the recombinant protein. The invention has the advantages of reducing the formation of inclusion bodies when the target protein is expressed in the periplasm space and improving the expression quantity.

Description

Culture medium and preparation method of escherichia coli exocytosis recombinant protein

Technical Field

The present invention relates to the field of recombinant protein production. More specifically, the invention relates to a culture medium and a preparation method of an extracellular secretion recombinant protein of escherichia coli.

Background

With the progress of genetic engineering technology, prokaryotic recombinant expression is an important means for obtaining a large amount of target recombinant protein at present. The technical route mainly comprises the following steps: obtaining target protein gene, subcloning the target protein gene, transferring the carrier carrying the target protein gene into a host cell for expression, and expressing the target recombinant protein by using proper culture conditions according to the characteristics of the carrier. The host cell widely applied to prokaryotic expression is mainly escherichia coli, and has the advantages that: the genetic background is clear, and the genetic modification is suitable; easy to culture, specific: the thalli reproduction speed is high; higher thallus density can be obtained; higher expression abundance can be obtained; the biological safety is good, the exogenous DNA transfection is easy, and the plasmid transfection efficiency is high.

Nevertheless, the deficiencies of E.coli as host bacteria still cause limitations on the expression of foreign genes. For example, proteins expressed in E.coli readily form inclusion bodies. In order to solve the problem of inclusion body formation, researchers have explored a variety of methods, including: 1. a weak promoter is adopted to reduce the expression level; 2. introducing molecular chaperones to increase the probability of correct folding of proteins; 3. the fusion tag is added on the premise of not influencing the biological activity of the target protein, so that the solubility of the target protein is improved; 4. using special host cells to make the intracellular environment in a non-reduction state; 5. periplasmic space expression. The establishment of these methods undoubtedly provides a larger application space for the expression of exogenous genes in Escherichia coli. However, although the target protein (recombinant protein) is expressed in the periplasmic space, some proteins still have inclusion bodies as the main final form.

How to reduce the formation of inclusion body when the target protein is expressed in periplasm space and improve the expression quantity is a problem which needs to be solved at present.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

It is still another object of the present invention to provide a medium which can reduce the formation of inclusion bodies when a target protein is expressed in a periplasmic space and increase the expression level.

It is still another object of the present invention to provide a method for preparing an extracellular secretion recombinant protein of E.coli, which can reduce the formation of inclusion bodies when the target protein is expressed in the periplasmic space and increase the expression level.

To achieve these objects and other advantages in accordance with the present invention, there is provided a medium comprising, in parts by weight: each liter of culture medium comprises 5-15g of triton, 12.5-50g of cane sugar, 5-15g of pyrophosphate and the balance of LB liquid culture medium.

Preferably, each liter of the culture medium comprises 10g of triton, 25g of sucrose, 10g of pyrophosphate and the balance of LB liquid culture medium.

Preferably, the pyrophosphate is at least one of potassium pyrophosphate and sodium pyrophosphate.

A preparation method of a live escherichia coli exocytosis recombinant protein comprises the following steps:

the method comprises the following steps:

s1, inoculating a seed solution containing an expression strain into an LB liquid culture medium to be cultured until the bacterial density OD is reached _600nm Adding Triton when the content is 1.8-2.2Introducing sucrose and IPTG, wherein the concentration of the IPTG is 50 mu mol/L;

s2, setting a culture temperature to be 25 ℃, adding pyrophosphate into the culture medium in the step S1, continuously culturing until fermentation is terminated, wherein the culture time is determined according to actual culture conditions for expression strains containing different genes, specifically, the activity of recombinant protein in fermentation supernatant can be cooperatively monitored in the culture process, and when the culture enters a plateau stage (the culture is slowly increased and is specifically judged according to actual conditions), the fermentation key point is reached, and the fermentation is terminated;

s3, taking the supernatant and purifying to obtain the recombinant protein.

Preferably, the preparation method of the seed liquid containing the expression strain specifically comprises the following steps: inoculating the expression strain into sterilized LB liquid culture medium at an inoculum size of 1% by volume, and culturing at 37 deg.C and 160rpm with shaking table to OD _600nm Is 1.8-2.2.

Preferably, in step S1, the seed solution containing the expressed strain is inoculated into LB liquid medium at an inoculum size of 5% by volume, and cultured at 37 ℃ and a shaker rotation speed of 160rpm to a cell density OD _600nm Is 1.8-2.2.

Preferably, the expression strain of step S1 is an expression strain of FAD-GDH, and step S2 is cultured in a shake flask for 16h.

The invention at least comprises the following beneficial effects:

1. prokaryotic cells are used for secretory expression, and have advantages over eukaryotic cells in the culture period;

2. the invention relates to a method for preparing inclusion body expression protein, which comprises the steps of adopting a secretory type carrier, wherein the carrier can secrete and express target protein into a periplasmic space, aiming at the problem that a large amount of protein is aggregated due to high local concentration of the protein entering the periplasmic space and an inclusion body is formed because of high synthesis speed. The key points of the main technology are as follows: a. the pyrophosphate has great promotion effect on the extracellular secretion expression of the escherichia coli; b. the pyrophosphate and the triton have the combined action, provide better dispersion effect on micro solid matters, ensure that the passage of cell walls and the outside is more smooth and the secretion effect is obviously improved.

3. Due to secretory expression, downstream purification is simple, purification can be performed only by collecting fermentation supernatant without breaking cells, the purification process is simplified, the purification difficulty is greatly reduced by less foreign proteins secreted into fermentation liquor, and a product with higher purity can be obtained through necessary purification.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis (SDS-PAGE) detection of a product according to one embodiment of the invention;

FIG. 2 is a polyacrylamide gel electrophoresis (SDS-PAGE) image of one of the products of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

1. Examples-media composition formulations, see table 1 in particular:

TABLE 1 examples recipe of Medium Components per liter

2. Taking prokaryotic expression platform with Escherichia coli as expression host to express flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) as an example:

a method for preparing an extracellular secretion recombinant protein of Escherichia coli by using the medium corresponding to examples 1 to 10, comprising the steps of:

s0, preparing seed liquid:

preparing an LB liquid culture medium, which specifically comprises the following steps: preparing LB liquid culture medium per liter, adding 10g of tryptone, 5g of yeast extract and 10g of NaCl into 950mL of deionized water, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using the deionized water;

subpackaging LB liquid culture medium according to the liquid loading amount of 1/5, placing in a high-pressure moist heat sterilization pot, adjusting the temperature to 121 ℃, and performing high-pressure moist heat sterilization for 20min;

preparation of expression strains for FAD-GDH: the FAD-GHD gene is derived from Aspergillus terreus NIH2624 with accession numbers on NCBI: XM _001216916; the gene sequence is optimized and constructed into a pET22b plasmid vector according to the codon preference of escherichia coli; escherichia coli host competent cells BL21 (DE 3) were purchased from Tiangen Biochemical technology (Beijing) Ltd, and plasmids were transformed into host cells BL21 (DE 3) to obtain expression strains of FAD-GDH, and they were preserved for future use;

inoculating the expression strain of FAD-GDH into sterilized LB liquid culture medium according to the inoculum size of 1% by volume ratio, and culturing at 37 deg.C and shaking table rotation speed of 160rpm to OD _600nm 1.8-2.2 to obtain seed liquid;

s1, inoculating a seed solution containing an expression strain into an LB liquid culture medium according to an inoculum size of 5% by volume, and culturing at 37 ℃ and a shaker rotating speed of 160rpm until the thallus concentration OD _600nm When the concentration is 1.8-2.2, correspondingly adding triton, sucrose and IPTG (isopropyl thiogalactoside subjected to filtration sterilization) according to the dosage in the tables 1 and 2, wherein the concentration of the IPTG is 50 mu mol/L;

s2, setting the culture temperature to be 25 ℃, adding corresponding pyrophosphate, continuing to culture for 16h, and terminating fermentation;

s3, taking the supernatant (fermentation supernatant) and purifying with nickel affinity filler to obtain the recombinant protein, wherein the specific method comprises the following steps:

s31, centrifuging the bacterial liquid after fermentation is stopped in the step S2 for 15min under the condition of a centrifugal speed of 10000g, and collecting supernatant (fermentation supernatant);

s32, filtering the fermentation supernatant by using a 0.45um composite cellulose filter membrane to obtain filtrate;

s33, adjusting the pH value of the filtered solution to 7.4 +/-0.1 by using NaOH/HCl;

s34, loading according to the proportion of 1KU (activity unit) of a nickel affinity column loading sample of 10 ml;

s35, regulating the flow rate of a peristaltic pump to 1/3 of the maximum linear flow rate of the filler, balancing at least 5 column volumes of the chromatographic column by using PBS buffer solution, and detecting the effluent by using a protein/nucleic acid ultraviolet detector until the baseline is completely leveled;

s36, after the sample loading is finished, washing the sample with PBS;

s37, washing impurities by using PBS (phosphate buffer solution) containing 50mM of imidazole respectively, and eluting the recombinant protein by using 300mM of imidazole after the impurity washing peak appears and drops to be flat, thereby obtaining the recombinant protein.

The formula of the components of the culture medium is shown in the table 2:

TABLE 1 comparative examples formula of culture Medium Components per liter

Number of
		Comparative example 1	LB liquid medium;
comparative example 2	10g of triton, 25g of cane sugar and the balance of LB liquid culture medium;
		comparative example 3	10g of triton, 25g of cane sugar, 10g of potassium polyphosphate and the balance of LB liquid culture medium;
comparative example 4	10g of triton, 25g of cane sugar and polyphosphoric acid10g of sodium and the balance of LB liquid culture medium;
		comparative example 5	25g of sucrose, 10g of potassium pyrophosphate and the balance of LB liquid culture medium;
comparative example 6	25g of sucrose, 10g of sodium pyrophosphate and the balance of LB liquid culture medium;

4. the screening of the culture medium, which is exemplified by the expression of flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) by a prokaryotic expression platform using Escherichia coli as an expression host, wherein the culture medium respectively corresponds to the culture medium described in examples 1-10 and comparative examples 1-8, comprises the following steps:

s0, preparing seed liquid:

s1, inoculating seed liquid containing an expression strain into an LB liquid culture medium according to an inoculation amount of 5% in volume ratio, and culturing at 37 ℃ and a shaking table rotating speed of 160rpm until the cell concentration OD is obtained _600nm At 1.8-2.2, triton, sucrose, and IPTG (isopropyl thiogalactoside filter sterilized) were added in the amounts of Table 1, table 2, examples and comparative examples, respectively, to form example 'and comparative example' corresponding to Table 3 below, and the concentration of IPTG was 50. Mu. Mol/L;

wherein, in comparative example 1, triton and sucrose are not added;

in comparative examples 5 and 6, triton was not added;

wherein the concentration of IPTG is 50 mu mol/L;

s2, setting the culture temperature to be 25 ℃, respectively performing the operations according to the following table 3, then continuing to culture for 16h, and terminating fermentation;

TABLE 3 correspondences between comparative examples and numbering

S3, respectively collecting the thalli and the supernatant (fermentation supernatant) of each example and each comparative example, crushing the thalli, and centrifuging to collect the supernatant (bacteria-crushing supernatant).

And (3) respectively carrying out viability detection on the fermentation supernatant and the broken bacteria supernatant, observing the viability of the FAD-GDH, wherein the detection results are shown in a table 4:

TABLE 4 viability of FAD-GDH in fermentation supernatants, cell disruption supernatants of examples and comparative examples

From table 3 above, it can be seen that:

(1) the content of triton in the culture media of examples 1', 2' and 3' is gradually increased, the activities of the corresponding FAD-GDH in the fermentation supernatant and the broken bacteria supernatant are increased and then reduced, and the activities of the FAD-GDH in the fermentation supernatant are far greater than those of the broken bacteria supernatant; the sucrose content in the culture media of examples 4', 2' and 5' is gradually increased, the activities of the corresponding FAD-GDH in the fermentation supernatant and the broken bacteria supernatant are increased and then decreased, and the activities of the FAD-GDH in the fermentation supernatant are far greater than those of the broken bacteria supernatant; the content of potassium pyrophosphate in the culture media of examples 6', 2' and 7' is gradually increased, the activities of the corresponding FAD-GDH in the fermentation supernatant and the bacteria-breaking supernatant are increased and then decreased, and the activities of the FAD-GDH in the fermentation supernatant are far greater than those of the bacteria-breaking supernatant; that is, when 10g of triton, 25g of sucrose and 10g of potassium pyrophosphate are contained in each liter of culture medium (example 2), the maximum value of the activity of FAD-GDH in the corresponding fermentation supernatant is 52.43, and the activity of FAD-GDH in the bacteria-breaking supernatant is only 2.18;

(2) examples 8', 9', 10' are characterized in that the content of sodium pyrophosphate in the culture medium is gradually increased, and the activities of the corresponding FAD-GDH in the fermentation supernatant and the broken bacteria supernatant are increased and then decreased, wherein when 10g of triton, 25g of sucrose and 10g of sodium pyrophosphate are contained in each liter of the culture medium (example 9), the activity of the corresponding FAD-GDH in the fermentation supernatant reaches a maximum value of 54.54, and the activity of the FAD-GDH in the broken bacteria supernatant is only 2.35;

(3) comparing comparative example 2 'with example 2' and example 9', it can be seen that the activity of FAD-GDH in the fermentation supernatant of comparative example 2' is lower than that of example 2 'and example 9', which indicates that the addition of pyrophosphate has a significant promoting effect on extracellular secretion expression of recombinant protein, and the mechanism is presumed to be: the pyrophosphate has good dispersion effect on micro solid matters, and the cells are not easy to agglomerate, so that the passage between the cell wall and the outside is more smooth, and the secretion effect is obviously improved;

furthermore, as can be seen from comparison of comparative example 2 'with comparative example 3' and comparative example 4', the viability of FAD-GDH in the fermentation supernatant of comparative example 2' is comparable to that of comparative example 3 'and comparative example 4', which indicates that although both polyphosphate and pyrophosphate belong to phosphate substances, the addition thereof has no significant promoting effect on extracellular secretion expression of recombinant protein;

(4) enzyme activity (activity of FAD-GDH) is not detected in both fermentation supernatant and broken bacteria supernatant corresponding to the comparative example 5', and by combining the results of example 2' and comparative example 2', it can be known that the addition of triton has no obvious promotion effect on extracellular secretion expression recombinant protein, and has combined action with potassium pyrophosphate, and the secretion effect is obviously improved by combining the two;

enzyme activity (activity of FAD-GDH) is not detected in both fermentation supernatant and broken bacteria supernatant corresponding to the comparative example 6', and by combining the results of example 2' and comparative example 2', it can be known that triton and potassium pyrophosphate have combined action, and the secretion effect is obviously improved by combining the two.

5. Polyacrylamide gel electrophoresis (SDS-PAGE) detection

The products of comparative example 1' and example 2 were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and detected as shown in FIGS. 1 and 2, respectively.

Wherein, in fig. 1, M corresponds to protein Marker, 1 is broken bacteria supernatant, 2 is broken bacteria precipitation, firstly, for the fermentation supernatant obtained in comparative example 1' has basically no detectable enzyme activity, further, fig. 1 shows that the broken bacteria supernatant has no obvious expression band, that is, after the conventional induction, the expression product is almost all inclusion body, therefore the expression vector is periplasm expression, and basically has no exocytosis under the conventional condition, wherein, the protein Marker in fig. 1 is purchased to beijing solibao science and technology company, and comprises 8 bands from top to bottom, the molecular weight is as shown in fig. 1;

in fig. 2, M corresponds to a protein Marker, 1 is a fermentation supernatant (step S31 is used for obtaining a sample), 2 is a column-loading flow-through (step S35 is used for obtaining), 3 is a impurity-washing part (step S37 is used for obtaining impurity-washing operation), and 4 is an elution part (recombinant protein), the graph shows that the fermentation supernatant contains the recombinant protein with a high proportion, the column-loading effect is good, the impurity protein is less in the impurity-washing process, and a high-purity product can be obtained in the elution process, which shows that due to secretion expression, downstream purification is simpler, and only the fermentation supernatant needs to be collected for purification without breaking cells, so that the purification process is simplified, and the difficulty of purification is greatly reduced because the impurity protein secreted into a fermentation broth is less: the protein Marker in fig. 2 was purchased from beijing solibao science and technology ltd, and includes 10 bands from top to bottom, and the molecular weight is shown in fig. 2.

While embodiments of the invention have been described above, it is not intended to be limited to the details shown, described and illustrated herein, but is to be accorded the widest scope consistent with the principles and novel features herein disclosed, and to such extent that such modifications are readily available to those skilled in the art, and it is not intended to be limited to the details shown and described herein without departing from the general concept as defined by the appended claims and their equivalents.

Claims

1. The preparation method of the escherichia coli exocytosis recombinant protein is characterized by utilizing a culture medium to prepare, wherein each liter of the culture medium comprises 5-15g of triton, 25-50g of sucrose, 5-12.5g of pyrophosphate and the balance of LB liquid culture medium, wherein the pyrophosphate is at least one of potassium pyrophosphate and sodium pyrophosphate;

the method for preparing the escherichia coli exocytosis recombinant protein by using the culture medium comprises the following steps:

s1, seeds containing expression strainsInoculating the solution into LB liquid culture medium, and culturing to obtain thallus concentration OD _600nm When the concentration is 1.8-2.2, adding triton, sucrose and IPTG, wherein the concentration of IPTG is 50 mu mol/L;

s2, setting the culture temperature to be 25 ℃, adding pyrophosphate into the culture medium in the step S1, and continuing to culture until fermentation is terminated;

s3, taking the supernatant and purifying to obtain the recombinant protein.

2. The method for preparing extracellular secretion recombinant protein of Escherichia coli according to claim 1, wherein each liter of the culture medium comprises 10g of triton, 25g of sucrose, 10g of pyrophosphate and the balance LB liquid medium.

3. The method for preparing the recombinant protein extracellularly secreted by Escherichia coli according to claim 1, wherein the method for preparing the seed solution containing the expressed strain comprises: inoculating the expression strain into sterilized LB liquid culture medium at an inoculum size of 1% by volume, and culturing at 37 deg.C and shaking table rotation speed of 160rpm to OD _600nm Is 1.8-2.2.

4. The method for preparing recombinant protein extracellularly secreted from Escherichia coli as claimed in claim 1, wherein in step S1, the seed solution containing the expressed strain is inoculated into LB liquid medium at an inoculum size of 5% by volume, and cultured at 37 ℃ and 160rpm with a shaker until the OD is reached _600nm Is 1.8-2.2.

5. The method for preparing extracellular secretion recombinant protein of Escherichia coli according to claim 1, wherein the expression strain of step S1 is an expression strain of FAD-GDH, and step S2 is performed by culturing in a shake flask for 16 hours.