High-efficiency expression and purification method of recombinant type IV collagen and application of recombinant type IV collagen in cosmetics

Abstract

The invention relates to the technical field of cosmetics, and particularly discloses a high-efficiency expression and purification method of recombinant type IV collagen and application of the recombinant type IV collagen in cosmetics, and the high-efficiency expression and purification method comprises the following steps: constructing an expression system; the method comprises the following steps of: selecting and treating a vector, inserting a target gene, transforming and screening host cells, regulating induced expression, purifying protein, performing primary treatment, performing affinity chromatography, performing fine purification and the like. Through fine optimization of an expression system, selecting pET-30b (+) plasmids with strong promoters, transferring the constructed recombinant plasmids into host cell escherichia coli, and performing primary treatment and affinity chromatography; the recombinant gene is subjected to codon optimization and other operations in a targeted manner, so that the transcription and translation of the recombinant four-type collagen gene are more efficient, and compared with the traditional unoptimized expression system, the protein expression quantity in a culture solution per unit volume can be increased by 45% or more, thereby providing a sufficient raw material basis for large-scale production. The ever-increasing demand of the market on the recombinant type 4 collagen is effectively met.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a method for efficiently expressing and purifying recombinant type IV collagen and an application thereof in cosmetics. Background Art

[0002] As a key component of the extracellular matrix, collagen is widely involved in many physiological processes, such as tissue structure support, cell adhesion, migration, and signal transduction. Among them, type IV collagen plays an indispensable role in the formation of basement membranes, and is of great significance in maintaining the normal morphology and function of tissues and organs. It has extremely critical application value in maintaining the homeostasis of tissues such as kidneys, blood vessels, and skin, and its demand in the fields of biomedicine, tissue engineering, and cosmetics is also increasing.

[0003] The traditional way to obtain type IV collagen mainly relies on natural tissue extraction. However, this method has many drawbacks that are difficult to overcome. On the one hand, the source of natural tissue is limited, and the collagen content in animal tissue is not high. Large-scale collection is not only costly, but also faces ethical controversy;

[0004] On the other hand, collagen extracted from natural tissues has low purity and complex impurities, including other types of collagen, glycoproteins, and various cell fragments. The subsequent complicated purification process will not only cause a large amount of protein loss, but also make it difficult to completely remove potential immunogenic substances, greatly limiting its direct clinical application.

[0005] With the vigorous development of genetic engineering technology, recombinant protein expression systems have emerged, opening up a new path for the acquisition of type IV collagen. At present, although there have been some studies on the expression and purification of recombinant type IV collagen, they are still in various difficulties. In the expression link, many existing technologies are difficult to achieve efficient expression, either because the gene sequence is not optimized for the host system, resulting in low transcription and translation efficiency; or the promoter activity of the expression vector used is insufficient to drive sufficient gene transcription, and the final recombinant protein yield is difficult to meet the market's ardent needs. In terms of purification, conventional purification methods have redundant steps and poor specificity, and often require multiple rounds of complex operations to obtain high-purity products. This not only greatly reduces the protein recovery rate, but also significantly increases the production cost, seriously hindering the industrialization process of recombinant type IV collagen. Summary of the invention

[0006] The object of the present invention is to provide a method for efficiently expressing and purifying recombinant type IV collagen and its application in cosmetics, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for efficiently expressing and purifying recombinant type IV collagen, S1, expression system construction vector selection and processing: pET-30b (+) plasmid is selected, the plasmid carries a kanamycin resistance gene, and Nde I and H i nd III are determined as restriction sites for the plasmid to accurately insert the recombinant type IV collagen gene;

[0009] Insertion of target gene: The 1041 bp recombinant type IV collagen gene fragment was connected to the pET-30b(+) plasmid double-digested with Nde I and Hind III by gene cloning technology.

[0010] Construct a recombinant plasmid and use the strong promoter on the plasmid to drive the subsequent efficient transcription of the recombinant type IV collagen gene in the host cell;

[0011] S2. Host cell transformation and screening

[0012] The constructed recombinant plasmid was transferred into the host cell Escherichia coli, and then the transformed cells were spread on a solid medium containing kanamycin and cultured overnight. A single colony was picked and verified by colony PCR and sequencing to ensure that the recombinant type IV collagen gene was integrated into the host cell genome, and the positive clones that successfully carried the recombinant plasmid were screened;

[0013] S3, Inducible Expression Regulation

[0014] Optimization of culture conditions: Inoculate positive clones into liquid culture medium containing kanamycin and culture at 37°C with shaking until the logarithmic growth phase;

[0015] Adding inducer: Adding inducer IPTG to the culture system to induce the expression of recombinant type IV collagen gene;

[0016] S4. Protein purification

[0017] Preliminary treatment: collect the bacterial solution after induced expression, collect the bacterial cells by centrifugation, break the cells by ultrasonic disruption to release the cell contents containing recombinant type IV collagen protein, then centrifuge to obtain the supernatant, and preliminarily separate the soluble protein and cell debris impurities;

[0018] Affinity chromatography: Since the recombinant protein carries a histidine tag (HHHHHH at the beginning of the sequence), a nickel ion affinity chromatography column was selected. The supernatant was loaded onto the affinity chromatography column. The recombinant protein was adsorbed due to the specific binding of the tag to nickel ions, and impurities flowed out. Then, the protein was gradient eluted with an elution buffer containing imidazole, and the elution peak containing recombinant type IV collagen was collected to improve the purity of the protein.

[0019] Fine purification: Ion exchange chromatography and gel filtration chromatography are used in series to further remove residual impurities based on the charge characteristics and molecular weight of recombinant type IV collagen, and gradually obtain high-purity recombinant type IV collagen with correct conformation, with a molecular weight of 34316.84 and a theoretical isoelectric point of 9.65.

[0020] Preferably, the target gene insertion is specifically to add a 1041 bp recombinant type IV collagen gene fragment and the above-mentioned double-enzyme-digested pET-30b(+) plasmid into a ligation reaction system at a molar ratio of 3:1, and then add DNA ligase and ligation buffer, connect at 16°C overnight to construct a recombinant plasmid.

[0021] Preferably, the host cell transformation step in S2 is to add the constructed recombinant plasmid to competent Escherichia coli (such as 100 μL of BL21 (DE3) competent cells precooled in an ice bath), gently mix, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and quickly return to the ice bath for 2 minutes. Add 900 μL of LB liquid medium preheated to 37°C, and shake and culture at 37°C and 200 rpm for 1 hour.

[0022] Preferably, the inducer adding step in S3 is to add IPTG to a final concentration of 0.5-1 mM, and continue shaking culture at 37° C. and 200 rpm for 4-6 hours to induce the expression of the recombinant type IV collagen gene.

[0023] Preferably, the recombinant type IV collagen is used in cosmetics.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses pET-30b (+) plasmid with a strong promoter through fine optimization of the expression system, and performs targeted codon optimization and other operations on the recombinant type IV collagen gene, so that the transcription and translation of the recombinant type IV collagen gene are more efficient. Compared with the traditional unoptimized expression system, the protein expression amount in the unit volume of culture medium can be increased by more than 1%, which provides a sufficient raw material basis for large-scale production and effectively meets the market's growing demand for type IV collagen. The purity of the recombinant type IV collagen finally obtained is improved, far exceeding the traditional purification method, greatly reducing the potential risk of impurities inducing immune response, making it more in line with the stringent standards in the field of biomedicine. And the mild induction expression conditions and precise purification parameter settings of the present invention can avoid protein denaturation and inactivation to the greatest extent. The obtained recombinant protein can accurately reproduce the biological function of natural type IV collagen in the extracellular matrix. When applied to scenes such as tissue repair and cell culture, it shows excellent effects of promoting cell adhesion, migration and proliferation, and ensures the reliability of the use effect. Moreover, compared with traditional methods, the method of the present invention can shorten the production cycle, speed up the product launch process, and respond more quickly to market dynamics and customer needs. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Embodiment 1:

[0028] 1. Experimental Materials and Reagents

[0029] Strains and plasmids: Escherichia coli strains (such as BL21(DE3)), pET-30b(+) plasmid;

[0030] Enzymes: NdeI, HindIII restriction endonucleases, DNA ligase;

[0031] Culture medium: LB solid medium and LB liquid medium, both with appropriate amount of kanamycin added;

[0032] Protein purification reagents: nickel ion affinity chromatography column and supporting buffer, imidazole, ion exchange chromatography column, gel filtration chromatography column and corresponding buffer;

[0033] Others: PCR related reagents, ultrasonic disruptor, etc.

[0034] 2. Construction of expression system

[0035] Vector selection and processing

[0036] Take out the pET-30b(+) plasmid, which comes with a kanamycin resistance gene for subsequent screening. Use NdeI and HindIII restriction endonucleases to perform double enzyme digestion on the plasmid. Reaction system (for example, a total volume of 50μL): 1μg pET-30b(+) plasmid, 1μL NdeI, 1μL HindIII, 5μL corresponding enzyme digestion buffer, add sterile water to 50μL. Incubate at 37℃ for 2-3 hours, then separate and purify the linearized plasmid after enzyme digestion by agarose gel electrophoresis.

[0037] Target gene insertion

[0038] A recombinant type IV collagen gene fragment with a length of 1041 bp was obtained, and it was added to the ligation reaction system with the above-mentioned double-enzyme-digested pET-30b(+) plasmid at a molar ratio of 3:1. Then, an appropriate amount of DNA ligase and ligation buffer were added, and the ligation was carried out at 16°C overnight to construct a recombinant plasmid.

[0039] 3. Host cell transformation and screening

[0040] Conversion

[0041] Add the constructed recombinant plasmid to competent E. coli (such as 100 μL of BL21 (DE3) competent cells pre-cooled in an ice bath), mix gently, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and quickly return to the ice bath for 2 minutes. Add 900 μL of LB liquid medium preheated to 37°C, and culture at 37°C, 200 rpm, shaking for 1 hour.

[0042] filter

[0043] Take an appropriate amount of the above culture and spread it on LB solid medium containing kanamycin (final concentration 50μg / mL) and culture it at 37℃ overnight. The next day, pick a single colony and inoculate it into a 96-well plate containing LB liquid medium containing kanamycin, 200μL per well, and shake and culture at 37℃ and 200rpm. Through colony PCR detection, the primers are designed for both ends of the recombinant type IV collagen gene, and the reaction program is set according to the requirements of conventional Taq enzyme. Select PCR positive clones for sequencing, and finally screen out positive clones carrying the correct recombinant plasmid.

[0044] IV. Inducible Expression Regulation

[0045] Optimization of culture conditions

[0046] The positive clones with correct sequencing were inoculated into a 250 mL conical flask containing 50 mL of LB liquid culture medium containing kanamycin (50 μg / mL), and cultured at 37°C and 200 rpm with shaking. Samples were taken every 1 hour to measure OD 600 , when OD600 When it reaches 0.6-0.8 (logarithmic growth phase), prepare for induction.

[0047] Inducer addition

[0048] IPTG was added to the culture system to a final concentration of 0.5-1 mM, and the culture was continued at 37° C. and 200 rpm for 4-6 hours to induce the expression of the recombinant type IV collagen gene.

[0049] 5. Protein Purification

[0050] Initial processing

[0051] Collect the bacterial solution after induced expression, centrifuge at 4°C, 8000rpm for 10 minutes to collect the bacteria. Resuspend the bacteria in pre-cooled PBS buffer, adjust the concentration of the bacteria, and use an ultrasonic disruptor to break the cells. The ultrasonic conditions are: work for 3 seconds, rest for 5 seconds, and the total time is 10-15 minutes. Centrifuge at 4°C, 12000rpm for 30 minutes, and take the supernatant.

[0052] Affinity chromatography

[0053] Slowly load the supernatant onto the equilibrated nickel ion affinity chromatography column at a flow rate of 1 mL / min. After loading, wash the column with a wash buffer containing 20 mM imidazole to remove non-specifically bound impurities until the effluent OD is 280 Then, the elution buffer containing 250 mM imidazole was used for gradient elution, and the eluate corresponding to the elution peak was collected.

[0054] Fine purification

[0055] The protein solution collected by affinity chromatography was loaded onto an ion exchange chromatography column. According to the charge characteristics of recombinant type IV collagen, a buffer with a suitable pH and salt concentration was selected for elution to collect the target protein peak. The collected solution was then loaded onto a gel filtration chromatography column. Based on its molecular weight of 34316.84, the impurities were further separated using the exclusion principle to finally obtain high-purity, conformationally correct recombinant type IV collagen. During the process, the pH of the buffer at each step was optimized with reference to the theoretical isoelectric point of 9.65.

[0056] Compared with traditional unoptimized expression systems, the protein expression level per unit volume of culture medium can be increased by more than 45%, providing sufficient raw material basis for large-scale production and effectively meeting the market's growing demand for recombinant type IV collagen.

[0057] The present invention is based on the recombinant plasmid constructed by pET-30b (+) plasmid, comprising a 1041bp gene fragment, which is inserted between NdeI and Hind III restriction sites and carries kanamycin resistance. The gene fragment encodes a protein composed of multiple repeated amino acid sequence units, the expected molecular weight of the protein encoded by the gene fragment is 34316.84, the theoretical isoelectric point of the protein encoded by the gene fragment is 9.65, and the nucleic acid sequence inserted in the plasmid, the amino acid sequence encoded by the nucleic acid sequence is a repetitive pattern, which is multiple

[0058] A series connection of the "GEFYFDLRLKGDKGVKGDKGNPGWPGAPGVPGPKGDPGFQGMPGIGGS" unit.

[0059] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0060] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for efficient expression and purification of recombinant type IV collagen, characterized in that: The following steps are involved: S1. Construction of expression system Vector selection and processing: pET-30b(+) plasmid was selected, which carries the kanamycin resistance gene. For this plasmid, Nde I and Hind III were determined as restriction sites for accurate insertion of the recombinant type IV collagen gene; Insertion of target gene: The 1041bp recombinant type IV collagen gene fragment was connected to the pET-30b(+) plasmid double-digested with Nde I and Hind III through gene cloning technology to construct a recombinant plasmid. The strong promoter on the plasmid was used to drive the subsequent efficient transcription of the recombinant type IV collagen gene in the host cell. S2. Host cell transformation and screening The constructed recombinant plasmid was transferred into the host cell Escherichia coli, and then the transformed cells were spread on a solid medium containing kanamycin and cultured overnight. A single colony was picked and verified by colony PCR and sequencing to ensure that the recombinant type IV collagen gene was integrated into the host cell genome, and the positive clones that successfully carried the recombinant plasmid were screened; S3, Inducible Expression Regulation Optimization of culture conditions: Inoculate positive clones into liquid culture medium containing kanamycin and culture at 37°C with shaking until the logarithmic growth phase; Adding inducer: Adding inducer IPTG to the culture system to induce the expression of recombinant type IV collagen gene; S4. Protein purification Preliminary treatment: collect the bacterial solution after induced expression, collect the bacterial cells by centrifugation, break the cells by ultrasonic disruption to release the cell contents containing recombinant type IV collagen, then centrifuge to obtain the supernatant, and preliminarily separate the soluble protein and cell debris impurities; Affinity chromatography: Since the recombinant protein carries a histidine tag (HHHHHH at the beginning of the sequence), a nickel ion affinity chromatography column was selected. The supernatant was loaded onto the affinity chromatography column. The recombinant protein was adsorbed due to the specific binding of the tag to nickel ions, and impurities flowed out. Then, the protein was gradient eluted with an elution buffer containing imidazole, and the elution peak containing recombinant type IV collagen was collected to improve the purity of the protein. Fine purification: Ion exchange chromatography and gel filtration chromatography are used in series to further remove residual impurities based on the charge characteristics and molecular weight of recombinant type IV collagen, and gradually obtain high-purity recombinant type IV collagen with a molecular weight of 34316.84 and a theoretical isoelectric point of 9.

65.

2. The method for efficient expression and purification of recombinant type IV collagen according to claim 1, characterized in that: The target gene insertion is specifically to add a 1041 bp recombinant type IV collagen gene fragment and the above-mentioned double-enzyme-digested pET-30b(+) plasmid to the ligation reaction system at a molar ratio of 3:1, then add DNA ligase and ligation buffer, and connect at 16°C overnight to construct a recombinant plasmid.

3. The method for efficient expression and purification of recombinant type IV collagen according to claim 1, characterized in that: The host cell transformation step in S2 is to add the constructed recombinant plasmid to the competent E. coli (such as 100 μL of BL21 (DE3) competent cells pre-cooled in an ice bath), gently mix, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and quickly return to the ice bath for 2 minutes. Add 900 μL of LB liquid medium preheated to 37°C, and shake and culture at 37°C and 200 rpm for 1 hour.

4. The method for efficient expression and purification of recombinant type IV collagen according to claim 1, characterized in that: The inducer addition step in S3 is to add IPTG to a final concentration of 0.5-1 mM, and continue shaking culture at 37° C. and 200 rpm for 4-6 hours to induce the expression of the recombinant type IV collagen gene.

5. The recombinant type IV collagen according to claim 1 is used in cosmetics.