CN114316065B - Small molecule peptide and application thereof, cell culture medium and nucleic acid molecule - Google Patents

Abstract

The invention belongs to the technical field of recombinant protein cell culture, and particularly relates to a small molecular peptide, application thereof, a cell culture medium and a nucleic acid molecule. The invention integrates the amino acid sequence of the cell membrane-penetrating peptide and the amino acid sequence of the functional structural domain of the autophagy regulatory protein to synthesize the small molecular peptide, and can promote autophagy of cells and realize high-efficiency expression of the recombinant protein in the cells by being used as a supplement additive of a mammalian cell culture medium for expression of the recombinant protein.

Description

Small molecule peptide and application thereof, cell culture medium and nucleic acid molecule

Technical Field

The invention belongs to the technical field of recombinant protein cell culture, and particularly relates to a small molecular peptide, application thereof, a cell culture medium and a nucleic acid molecule.

Background

Mammalian cell expression systems have been widely used for the production of recombinant protein drugs due to their ability to make complex and physiologically relevant post-translational modifications. Chinese hamster ovary (Chinese hamster ovary cells, CHO) cells have become the most common cell line for the industrial production of recombinant glycoproteins due to their own nature and their ability to produce human-like glycosylation. Over 90% of approved marketed antibody drugs are currently produced in mammalian cells. For many years, a great deal of work has been done around increasing the expression level of recombinant proteins in CHO cells, and advances have been made in the optimization of medium formulation, culture conditions, and cell lines.

Apoptosis caused by nutrient deficiency is a common cell death mechanism affecting biological processes. Autophagy is a process of phagocytizing and coating self cytoplasmic proteins or organelles into vesicles and fusing with lysosomes to form autophagy lysosomes, degrading the contents of which it is coated, thereby achieving metabolic needs of the cells themselves and renewal of certain organelles. Autophagy is often a critical turning point between cell survival and death. Autophagy is a key relieving response to sources of new metabolic stress, including nutritional deterioration, through vacuolation, degradation and circulation of large protein complexes to the entire organelle, thereby maintaining the energy requirements and homeostasis of the cell. The recombinant protein expression level of CHO cells can be increased by the addition of media with autophagy-inducing related chemicals such as 3-methyladenine (3-MA), dorsomorphin (ATP competitive AMPK inhibitor), SP 600125. Although the addition of chemicals in cell culture to induce autophagy to enhance CHO cell recombinant protein expression is feasible, their use is accompanied by a risk of confounding effects (Wu et al Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on Class I and III Phosphoinositide 3-Kinase. Journal of Biological Chemistry,285 (14), 10850-10861). Therefore, preference should be given to using a lower concentration, more specific replacement reagent for addition.

The components of the serum-free culture medium for mammalian cells commonly used at present contain plant hydrolysates such as yeast hydrolysates, and the hydrolysates contain various small molecular peptides to promote cell growth and increase expression level, and some researches exist to adopt small peptides comprising 2-6 amino acids as amino acid supplements of the cell culture medium to solve the problems of instability/precipitation of the amino acid components supporting the maximum cell growth and/or protein or virus production, such as patent CN106520658A, but no small molecular peptide inducing autophagy is used for expression of cell recombinant proteins at present.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a small molecular peptide which is taken as a functional component of a mammalian cell culture medium, can promote and induce autophagy, and can improve the expression quantity of cell recombinant proteins.

It is a second object of the present invention to provide nucleic acid molecules capable of encoding the small molecule peptides of the present invention.

The third object of the present invention is to provide the application of the small molecule peptide of the present invention in culturing mammalian cells, and to increase the expression level of recombinant protein in mammalian cells as a supplement to the culture medium.

The fourth object of the present invention is to provide a cell culture medium containing the small molecule peptide of the present invention.

In order to achieve the above object, the present invention adopts the following technical scheme:

a small molecular peptide is prepared from cell-penetrating peptide and small peptide with autophagy regulatory protein function.

Preferably, the C-terminal of the cell-penetrating peptide is covalently linked to the N-terminal of a small peptide having autophagy modulating protein function; further, the cell penetrating peptide is covalently linked to a small peptide having the function of an autophagy controlling protein by two amino acid molecules.

It should be noted that the "small molecule peptide" of the present invention refers to a linear molecule formed by connecting amino acid residues through peptide bonds; the cell penetrating peptide is a short peptide capable of carrying macromolecular substances into cells, is rich in basic amino acid residues such as arginine, lysine and the like, and can be used for improving and improving the performance of the cell penetrating peptide by mutating or modifying the amino acid sequence of the short peptide; the "small peptide with autophagy regulatory protein function" is preferably a small peptide designed according to the amino acid sequence of the autophagy regulatory protein function determining domain, more preferably 267-284 amino acid sequence of autophagy key regulatory protein complex-Beclin 1 (GenBank: AAD 27650.1), and it is further understood that amino acid sequences formed by substitution of functionally similar homopolar amino acids without affecting autophagy regulatory function can be applied to the present invention.

In some embodiments of the invention, the amino acid sequence of the small peptide of autophagy modulating protein function is TNVFNATFEIWHDGEFGT, as shown in SEQ ID No. 1; the amino acid sequence of the cell penetrating peptide is YGRKRRQRRRR, and is shown as SEQ ID NO. 2; or YGRKKRRQR as shown in SEQ ID NO. 3; or GRKKRRQRRRPPQC as shown in SEQ ID NO. 4.

Specifically, the amino acid sequence of the small molecule peptide is YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT, and is shown as SEQ ID NO. 5; or YGRKKRRQRGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 6; or GRKKRRQRRRPPQCGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 7.

The small molecule peptides of the invention may be synthesized by conventional solid phase synthesis methods known to those skilled in the art. For example, the small peptide of the invention can be synthesized by a Applied Biosystem synthesizer or a Pioneer peptide synthesizer according to solid-phase chemical technology, and the steps are generally that a first amino acid is combined with an amino acid protecting agent and resin, then the first amino acid protecting group is removed, then a condensing reagent is added to combine with a second amino acid, the same operation is carried out until the last amino acid is combined, and the small peptide is obtained after HPLC purification.

The invention also provides a nucleic acid molecule which can code the small molecule peptide, and the small molecule peptide can be prepared by a way of transfection and expression of a nucleic acid molecule connection carrier.

A cell culture medium comprising the above small molecule peptide. Other conventional media components such as carbohydrates, inorganic salts, trace elements, growth factors, buffers, and the like are also included; can be prepared by adding the small molecule peptide to any cell culture commercially available, preferably by adding the small molecule peptide to a serum-free medium.

In some embodiments of the invention, the small molecule peptides described above are used in cell culture processes for recombinant protein expression; wherein the cell is preferably a mammalian cell, optionally a CHO cell, COS cell, VERO cell, BHK cell, AE-1 cell, SP2/0 cell, L5.1 cell, perC6, hybridoma cell or HEK 293, preferably a CHO cell, further the CHO cell may be CHO-S, CHO-K1 or CHO-DG44. Preferably, the small molecule peptide is added to a recombinant CHO cell stable strain containing a target gene during suspension serum-free culture; specifically, the final concentration of the small molecule peptide is 1.0 mu mol/L to 5 mu mol/L.

The specific production method of the small molecule peptide applied to CHO cell recombinant protein expression comprises the following steps:

(1) Carrying out suspension serum-free culture on a recombinant CHO cell stable strain containing a target gene;

(2) Adding a small molecule peptide;

(3) Culturing at 37deg.C to logarithmic phase, and culturing at 33deg.C;

(4) The cell activity was reduced to below 50%, and the culture supernatant was collected and examined for expression of recombinant proteins.

The small molecule peptide structure comprises a cell membrane-penetrating peptide amino acid sequence and an amino acid sequence with autophagy regulatory protein function, and can promote cell autophagy by being used as a supplement additive of a recombinant protein expression culture medium, so that the high-efficiency expression of the recombinant protein in cells is realized.

Drawings

FIG. 1 is a graph showing the effect of small molecule peptides of varying concentration and molecular structure on CHO cell growth density;

FIG. 2 shows the effect of small molecule peptides of varying concentration and molecular structure on CHO cell activity;

FIG. 3 is the effect of small molecule peptides of varying concentration and molecular structure on CHO cell recombinant albumin;

FIG. 4 shows the effect of different temperature culture conditions on CHO cell growth density;

FIG. 5 shows the effect of different temperature culture conditions on CHO cell activity;

FIG. 6 shows the effect of different temperature culture conditions on recombinant albumin of CHO cells.

Detailed Description

The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; the culture medium, the reagent, the cell line reagent and the like used in the examples and the test examples are commercially available.

Used in the following examples and test examplesThe stable CHO cell line is a CHO cell line of human HSA constructed according to conventional methods known in the art, cells were passaged every 3d, at CHO ProGrowth in serum-free medium.

The small molecule peptides of the following examples are prepared by combining a first amino acid with an amino acid protecting agent and resin, removing the first amino acid protecting group, adding a condensing agent to combine with a second amino acid, performing the same operation until the last amino acid is combined, and purifying by HPLC.

Example 1

This example provides a small molecule peptide having the amino acid sequence of

YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT, as shown in SEQ ID NO. 5.

Example 2

The amino acid sequence of the small molecule peptide provided in this example is

YGRKKRRQRGGTNVFNATFEIWHDGEFGT, as shown in SEQ ID NO. 6.

Example 3

The amino acid sequence of the small molecule peptide provided in this example is

GRKKRRQRRRPPQCGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 7.

Comparative example

The amino acid sequence of the small molecule peptide provided in this comparative example is

YGRKKRRQRRRGGVGNDFFINHETTGFATEW as shown in SEQ ID NO. 8.

The small molecule peptide amino acid sequence provided in this comparative example comprises the same cell penetrating peptide domain amino acid sequence as in example 1, but the amino acid sequence of this example linked to the cell penetrating peptide amino acid sequence is an ineffective Beclin 1 amino acid sequence, and does not have autophagy inducing function.

Experimental example 1 Effect of small molecule peptides on expression of recombinant proteins by CHO cells

Test materials: the small molecule peptides provided in examples 1 to 4 above

The test method comprises the following steps: the CHO cell strain of human HSA is cultured, small molecular peptide is added during the culture process, and the specific culture method comprises the steps of initial cell quantity of 5-6 multiplied by 10 ⁶ mu.l/mL, 30mL CHO Pro was addedAdding small molecular peptide into the culture medium, performing suspension culture at 37 ℃ at 120rpm, taking 300 μl of sample every 2 days, and monitoring cell density, cell activity, cell diameter and cell aggregation;

density reaches 2 x 10 ⁶ Mu.l of the culture broth were mixed with 200. Mu.l of 1 XDulbecco's phosphate buffer (DPBS) at a volume of 1:3 were subjected to cell count and viability assays. The remaining culture was centrifuged to remove cells, about 150. Mu.L of supernatant was retained, albumin production was measured by ELISA, and the experiment was repeated three times.

Test grouping: a total of A, B, C, D, E, F, G seven parallel test groups were assigned to different groups, wherein group A was charged with 1.0. Mu. Mol/L of example 1 small peptide, group B was charged with 2.0. Mu. Mol/L of example 1 small peptide, group C was charged with 3.0. Mu. Mol/L of example 1 small peptide, group D was charged with 4.0. Mu. Mol/L of example 1 small peptide, group E was charged with 5.0. Mu. Mol/L of example 1 small peptide, group F was blank control group without small peptide, group G was charged with 2.5. Mu. Mol/L of comparative small peptide.

Test results:

the results of monitoring the cell density and cell activity of the different test groups are shown in fig. 1 and 2, and the results of measuring the volume expression amount of HSA protein of the different test groups are shown in fig. 3:

there was no significant difference in cell density and cell activity among the groups of cells. A. The volume expression amounts of the five groups of B, C, D, E HSA are 361.14mg/L, 379.24mg/L, 381.34mg/L, 407.31mg/L, 375.39mg/L, 211.03mg/L and 216.12mg/L respectively, which are higher than those of the blank group F and the group G added with the small molecular peptide of the comparative example.

The same test method as described above was used to verify that the amount of protein expressed by adding the small molecule peptides provided in examples 2 and 3 of the present invention was substantially equivalent to that of example 1;

according to the test result, the cell membrane-penetrating peptide amino acid sequence and the autophagy regulatory protein functional domain amino acid sequence are integrated to synthesize the small molecule peptide, so that the cell autophagy can be induced, and the recombinant protein expression quantity of the CHO cell is improved.

Experimental example 2 Effect of Low temperature on expression of recombinant proteins by CHO cells

The test examples were recorded in groups H

The test method comprises the following steps:

the method for culturing the CHO cell strain of the human HSA comprises the steps of adding small molecule peptide during the culturing process, wherein the specific culturing method comprises the steps of adding 30mL CHO Pro into the CHO cell strain containing HSA encoding genes with initial cell quantity of 5-6 multiplied by 106/mL Serum-free culture medium, adding 4.0 mu mol/L small molecule peptide into the culture medium, suspending and culturing at 37deg.C and 120rpm, culturing to logarithmic phase (37 deg.C to fourth day), and culturing at 33deg.C; monitoring cell density, cell viability, cell diameter and cell aggregation during the culture;

the density reached 2X 106/ml, 100. Mu.l of the culture broth was mixed with 200. Mu.l of 1 XDulbecco's phosphate buffer (DPBS) to give a density of 1:3 were subjected to cell count and viability assays. The remaining culture was centrifuged to remove cells, about 150. Mu.L of supernatant was retained, albumin production was measured by ELISA, and the experiment was repeated three times

Test results:

the cell density, cell activity, and HSA volume expression level of the methods of comparative examples 6 and 8 are shown in FIGS. 4 to 6

The results of monitoring the cell density and cell activity of the group D and the group H are shown in fig. 4 and 5, and the results of detecting the volume expression amount of HSA proteins of the group D and the group H are shown in fig. 6:

the cell densities of the D group and the H group are slightly higher by changing the culture temperature, and the cell activities of the groups are not significantly different. The volume expression quantity of HSA of the group D and the H is 406.21mg/L and 576.04mg/L respectively, and the expression quantity of the group H is 1.42 times of that of the group D;

from the results, the small molecular peptide provided by the invention can further improve the protein expression under the low-temperature culture condition.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will appreciate that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features can be replaced equivalently; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims (4)

1. The application of the small molecular peptide as the cell culture medium additive is characterized in that the small molecular peptide is prepared by connecting cell membrane-penetrating peptide with small peptide with autophagy regulatory protein function;

the cell culture is CHO cell culture; the amino acid sequence of the small molecule peptide is YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT, and is shown as SEQ ID NO. 5;

or YGRKKRRQRGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 6;

or GRKKRRQRRRPPQCGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 7.

2. The use of claim 1, wherein the small molecule peptide is added to a serum-free medium to prepare a cell culture medium for cell culture.

3. The application of the small molecular peptide in the aspect of cell culture of recombinant protein expression is characterized in that the small molecular peptide is used as a cell culture medium additive and applied to CHO cell culture, and the amino acid sequence of the small molecular peptide is YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 5;

or YGRKKRRQRGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 6;

or GRKKRRQRRRPPQCGGTNVFNATFEIWHDGEFGT as shown in SEQ ID NO. 7.

4. The use according to claim 3, wherein the small molecule peptide is added when a recombinant CHO cell stable strain containing the gene of interest is subjected to suspension serum-free culture; adding the micromolecular peptide with the final concentration of 1.0 mu mol/L to 5 mu mol/L; the specific production method comprises the following operation steps:

(1) Carrying out suspension serum-free culture on a recombinant CHO cell stable strain containing a target gene;

(2) Adding a small molecule peptide;

(3) Culturing at 37deg.C to logarithmic phase, and culturing at 33deg.C;

(4) The cell activity was reduced to below 50%, and the culture supernatant was collected and examined for expression of recombinant proteins.