A recombinant humanized type I collagen protein expressed and purified in Escherichia coli and its application

Abstract

The present invention discloses a recombinant humanized type I collagen protein based on expression and purification in Escherichia coli and its application, belonging to the field of genetic engineering technology. The recombinant humanized type I collagen protein prepared by the present invention includes a basic repeating unit, and the amino acid sequence of the basic repeating unit is GTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSP; in addition, according to the codon preference of Escherichia coli, the optimized codon sequence is shown in SEQ ID NO:4, and has a translation pause site, which is conducive to the folding of the target protein. The type I collagen protein of the present invention has high biological activity, can promote the adhesion and proliferation of human fibroblasts, repair the skin barrier, and can be widely used in the fields of cosmetics and medical and medical products.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant humanized type I collagen protein expressed and purified in Escherichia coli and an application thereof. Background Art

[0002] Collagen is one of the most important and abundant proteins in mammals. It is a structural protein found in human skin, connective tissue and bones, as well as other tissues. Type I collagen plays an important role in the regulation of the organization structure and function of the extracellular matrix, and is involved in many physiological and pathological processes, including structural support, cell adhesion and differentiation, inflammation-related pathological processes, and association with certain diseases. Collagen is biodegradable, has good biocompatibility, and low immunogenicity; it can promote cell proliferation and adhesion; in addition, collagen can form collagen fibers and has certain mechanical properties. It is a good biomaterial that can be widely used.

[0003] Extracting collagen from animal tissues has serious problems such as difficulty, low purity, and high cost, and the batch-to-batch stability of collagen is very poor due to differences in individual animals. The applicant and inventor's prior application CN118994374A discloses a recombinant humanized type XVII collagen and a preparation method thereof, and CN117986355A discloses a recombinant humanized type III collagen and a preparation method and use thereof, etc., which all obtain recombinant humanized collagen by genetic engineering methods and have good biological activity. It can be seen that optimizing collagen with a specific amino acid sequence by means of genetic engineering has obvious advantages.

[0004] Collagen prepared by recombinant expression has the advantages of single molecular weight, high purity, and no hidden dangers of virus transmission. Therefore, it has begun to be used to replace animal collagen in the fields of cosmetics or pharmaceutical products. Recombinant collagen is constructed by genetic engineering technology to express engineered bacteria containing the full-length gene or partial fragment gene of collagen, and then synthesize collagen through engineered bacteria, and finally use a specific purification process to obtain high-purity recombinant collagen. The performance of human-like collagen produced by genetic engineering is similar to that of natural collagen, but compared with natural collagen, human-like collagen has no hidden dangers of viruses and low immune rejection reactions. It not only retains the original efficacy of collagen, but also gives it new functions, such as processability, water solubility, quality controllability, etc. Therefore, the use of genetic engineering technology to construct genetically engineered strains is of great significance for the preparation of high-quality human-like collagen. Summary of the invention

[0005] The technical problem to be solved by the present invention is to utilize genetic engineering technology to recombinantly express a new humanized type I collagen and an expression and preparation method of the recombinant type I collagen based on a prokaryotic expression vector Escherichia coli.

[0006] Based on the above, the technical solution of the present invention is achieved as follows:

[0007] The present invention provides a recombinant humanized type I collagen. The amino acid sequence of the recombinant humanized type I collagen comprises a basic repeating unit. The amino acid sequence of the basic repeating unit is shown in SEQ ID NO: 1: GTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSP.

[0008] Furthermore, the amino acid sequence of the recombinant humanized type I collagen is shown as (GTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSP)n, where n≥1 and is an integer.

[0009] Furthermore, the value of n is 8-20; preferably 12.

[0010] Furthermore, the amino acid sequence of the recombinant humanized type I collagen is as shown in SEQ ID NO:2.

[0011] Furthermore, the amino acid sequence of the recombinant humanized type I collagen has at least 80% sequence identity with SEQ ID NO: 2, specifically can be any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% or a range between any two of them.

[0012] Furthermore, the amino acid sequence of the recombinant humanized type I collagen may have one or more amino acid substitutions, deletions, insertions and / or additions based on the sequence shown in SEQ ID NO: 2. Optionally, the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID No. 2, 1, 2 or 3 amino acids are replaced by amino acids with similar or similar properties to form a peptide, and the function of the collagen is not changed after the substitution.

[0013] The present invention also provides a nucleic acid molecule encoding the recombinant humanized type I collagen.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO:3-4.

[0015] The invention also provides a recombinant expression vector comprising the nucleic acid molecule.

[0016] Furthermore, the recombinant expression vector is based on pET-28a.

[0017] The present invention also provides a recombinant bacterium of the recombinant humanized type I collagen.

[0018] Furthermore, the recombinant bacteria uses Escherichia coli BL21 (DE3) as a host.

[0019] It should be noted that recombinant bacteria refers to any cell type, which is susceptible to transformation, transfection, transduction, etc. of a nucleic acid construct or expression vector comprising a polynucleotide of the present invention." Recombinant bacteria" covers any offspring of a parent cell, which is not identical to the parent cell due to mutations during the replication process. The host cell can be any cell useful in the production of recombinant humanized collagen of the present invention. In order to produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be separated and inserted into one or more vectors to further clone and / or express in the host cell. Conventional techniques (for example, by using oligonucleotide probes that can specifically bind to genes encoding recombinant collagen) can be used to easily separate and sequence such nucleic acids. The host cell refers to a cell into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, regardless of the number of passages. Offspring may not be identical to parent cells in nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well known, for example, using electroporation to introduce vectors into host cells, and the method may also be transfection, microinjection technology, gene gun technology, liposome-mediated method, etc. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli.

[0020] The present invention also provides a method for producing the recombinant humanized type I collagen, comprising the following steps: inoculating a recombinant bacterium expressing the recombinant humanized type I collagen into a fermentation medium at an inoculation rate of 8-12%, fermenting and culturing for 30-40 hours, breaking the bacterial body and centrifuging to obtain a supernatant containing the recombinant humanized type I collagen, and purifying and desalting to obtain the type I collagen.

[0021] Furthermore, the purification method is selected from any one or more of salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.

[0022] The present invention also provides a composition, which includes the recombinant type I collagen, or the recombinant type I collagen encoded by the nucleic acid molecule, or the humanized type I collagen expressed by the recombinant expression vector, or the humanized type I collagen produced by the host cell, or the humanized type I collagen prepared by the production method.

[0023] The present invention also provides application of the composition in preparing cosmetics or medical device products.

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

[0025] The invention provides a novel humanized type I collagen protein. A humanized type I collagen protein gene is designed based on a characteristic collagen protein sequence Gly-XY. Meanwhile, the nucleotide sequence is optimized according to the preference of Escherichia coli, and more translation pause sites appear in the optimized codons. The constructed expression plasmid is introduced into Escherichia coli BL21 (DE3). The type I human-like collagen protein of the invention has good hydrophilicity and a higher expression amount, and can be assembled at will to form a humanized type I collagen protein with a large molecular weight.

[0026] The recombinant type I collagen prepared by fermentation using Escherichia coli as the expression host does not contain endotoxin, has low production cost, high protein expression, stable quality, and solves the problem of immunogenicity. The type I collagen of the present invention has high biological activity, can promote the adhesion and proliferation of human fibroblasts, repair the skin barrier, and can be widely used in the fields of cosmetics and pharmaceutical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Translation curve of the recombinant humanized type I collagen of the present invention (the wavy line is the blue line, and the middle horizontal line is the red line); wherein A is SEQ ID NO: 3 before optimization, and B is SEQ ID NO: 4 after optimization.

[0029] Figure 2 The cytotoxicity of the recombinant humanized type I collagen of the present invention;

[0030] Figure 3 The cell proliferation promoting activity of the recombinant humanized type I collagen of the present invention;

[0031] Figure 4 The cell adhesion promoting activity of the recombinant humanized type I collagen of the present invention. DETAILED DESCRIPTION

[0032] The present invention is described below by way of examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the medicinal materials and reagents described can be obtained from commercial channels unless otherwise specified; and the performance of products from different sources does not have a significant impact.

[0033] For those skilled in the art, the features, benefits and advantages of the present invention will become apparent by reading the contents disclosed in this specification.

[0034] Unless otherwise indicated, all percentages, fractions and ratios are calculated based on the total weight of the composition of the present invention. The term "mass content" herein may be represented by the symbol "%".

[0035] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention may comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0036] Example 1 Gene design and synthesis

[0037] The amino acid sequence of recombinant humanized type I collagen is SEQ ID NO: 2:

[0038] GTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSPGTRGADGPAGPSGISGMPGPPGPGGDQGSPGPPGSP。

[0039] The nucleotide sequence of recombinant humanized type I collagen is SEQ ID NO:3:

[0040]

[0041] The gene sequence was reversely designed using the online codon optimization tool (ExpOptimizer) (https: / / www.novopro.cn / tools / codon-optimization.html). The NdeI and XhoI restriction sites were removed during the design process for the preferred codons required for expression in the host Escherichia coli, which was beneficial for later gene manipulation. The optimized gene sequence is shown in SEQ ID NO:3. Compared with the original gene sequence, the GC content of this sequence decreased from 86.11% to 64.43%, which is more conducive to the efficient expression of the gene in the host bacteria Escherichia coli.

[0042] The optimized gene sequence of recombinant humanized type I collagen is SEQ ID NO: 4 as shown below:

[0043]

[0044] The translation pause curves of type I collagen protein before and after codon optimization calculated by RiboTempo software are as follows: Figure 1 As shown in A (before optimization) and B (after optimization), the red line of the translation pause curve forms a translation pause site in the predetermined area (the red line is lower than the blue line). The translation speed of the unoptimized nucleotide base of SEQ ID NO.3 on the ribosome is relatively constant, and there is no reduction in the translation speed. However, during the translation of the optimized SEQ ID NO:4 on the ribosome, the translation speed will be significantly reduced throughout the entire process, and more translation pause sites will appear (the red line is lower than the blue line), so that the translated fibronectin can have enough time to fold, so that a collagen product with higher biological activity and better expression can be obtained.

[0045] According to the gene sequence shown in SEQ ID NO: 4, the nucleotide fragment of 1296 bases was handed over to Shanghai Biotechnology for whole gene synthesis.

[0046] Example 2 Construction, expression and purification of expression vector

[0047] (1) The DNA fragment of SEQ ID NO:4 was cloned and amplified by PCR, and the target gene was recovered by agarose gel electrophoresis. The NdeI and XhoI restriction sites added to the primers were used to add the corresponding restriction endonucleases to cut the gene fragment into sticky ends, and the gene fragment was co-incubated with the pET-28a expression vector treated with the corresponding restriction endonucleases, and T4 DNA ligase was added to connect the fragments at 4°C for 12 hours, and then transformed into Escherichia coli DH5α; the white spot clones were selected by ampicillin sodium-LB plate screening, and the plasmids were extracted by alkaline lysis or boiling method of molecular cloning, and the positive clones were identified by restriction endonuclease and PCR methods. Finally, sequencing was performed to confirm that the recombinant plasmid contained the correct gene sequence reading frame, and the pET-28a-1296 recombinant expression vector was successfully constructed.

[0048] (2) Transform the correct pET-28a-1296 plasmid into E. coli BL21 (DE3), screen for single-spot clones using ampicillin sodium-LB plates, culture overnight in 5 ml LB medium, transfer at a ratio of 1:100, and culture in a shake flask at 37°C until OD 600 The concentration of the purified protein was 0.4-0.6, and IPTG stock solution was added at a dilution of 1:5000. The cells were cultured at 20°C for 8-10 hours, and the cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next purification step.

[0049] (3) Wash the mixed bacterial precipitate with PBS buffer, resuspend about 500 ml of bacterial solution in 20-40 ml volume, use lysozyme and Triton X-100 to help lyse the bacteria, ultrasonically break the bacteria in an ice-water mixture environment for 45 minutes, centrifuge at 12000 rpm / min for 20 minutes, and collect the supernatant. At this time, the solution contains a large amount of target type I collagen.

[0050] (4) Select ammonium sulfate precipitation and graded salting out for preliminary purification. After exploring the saturation of ammonium sulfate, the optimal saturation of ammonium sulfate salting out is 20%. Use PBS buffer to wash the nickel ion affinity column (Ni-NTA His-Bind Resin) column material, then mix the column material and the above desalted and purified solution and co-incubate at room temperature or on ice for 30 minutes, then load the column, wash the impurities with PBS solution containing 15mM imidazole, and only pure type I collagen will remain on the column. Add an appropriate amount of Prescission Protease with His tag to the column, shake it at room temperature or on ice for 2 hours, and the type I collagen protein can be released from the column. Elute with PBS solution to obtain recombinant humanized type I collagen of SEQ ID NO: 2 with a purity of more than 95%, and freeze-dry it into dry powder for later use.

[0051] Example 3 Evaluation of cytotoxicity of recombinant humanized type I collagen

[0052] Take HeLa cells that have grown to 70%-80% of the bottom area of ​​the culture flask, digest them with 0.25% trypsin, and use complete culture medium to make the cell density of 1×10 5 / mL of cell suspension. Take 100μL of cell suspension and inoculate it in a 96-well culture plate and culture it in an incubator at 37℃ and 5% CO2 saturated humidity. After 24h of cell culture, aspirate the complete culture medium. Add recombinant humanized type I collagen solution diluted with high-glucose DMEM medium to the experimental group at a concentration of 0.05mg / ml and 0.10mg / ml (3 replicates for each concentration), the control group is cells cultured in DMEM medium, and the blank group does not contain collagen. Continue to culture in an incubator at 37℃ and 5% CO2 saturated humidity for 24h. Add 10μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 4h, and use an enzyme-linked immunosorbent assay to measure the absorbance value (OD value) of each well at a wavelength of 450nm. Calculate the cell survival rate according to the mean absorbance of each group according to the following formula:

[0053] ;

[0054] In this example, the control group was a commercially available recombinant type I collagen sample solution, which was added into the cell culture medium at a concentration of 0.10 mg / ml, with 3 parallel samples in each group.

[0055] The experimental results are as follows Figure 2 As shown, the cell viability of each experimental group was above 100%, indicating that the recombinant humanized type I collagen prepared by the present invention had no cytotoxicity and good safety.

[0056] Example 4 Evaluation of the cell proliferation promoting activity of recombinant humanized type I collagen

[0057] BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were selected and inoculated into 96-well cell culture plates. After digestion with 0.25% trypsin, the cells were centrifuged and resuspended in DMEM medium containing 10% fetal bovine serum and counted. BALB / c 3T3 cells were inoculated into 96-well culture plates, with the number of cells per well being approximately 5×10 3 , and set up a blank control without cells, and cultured overnight at 37°C, 5% CO2. Recombinant humanized type I collagen prepared in Example 2 was added at a final concentration of 0.05 mg / ml and 0.10 mg / ml, respectively, with 3 replicates per group, and cultured at 37°C, 5% CO2 for 48 hours. 4 hours before the end of the culture, 5 g / L of MTT solution was added, and the culture was terminated after another 4 hours of culture; the liquid in the well was discarded, washed once with PBS, DMSO was added to dissolve the crystals, and the absorbance value of each well was measured at a wavelength of 570 nm.

[0058] Cell proliferation rate = (absorbance value of the assay well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100%.

[0059] In this example, the control group was a commercially available recombinant type I collagen sample solution, which was added into the cell culture medium at a concentration of 0.10 mg / ml, with 3 parallel samples in each group.

[0060] like Figure 3 As shown, the experimental results show that under the condition of a concentration of 0.10 mg / ml, the proliferation rates of the collagen in the control group and the recombinant humanized type I collagen of the present invention on BALB / c 3T3 cells are 35.2±6.2% and 40.6±8.5%, respectively, and both are better than 17.5±5.2% of the recombinant humanized type I collagen of the present invention at a concentration of 0.05 mg / ml, indicating that the recombinant humanized type I collagen prepared by the present invention can significantly promote the proliferation of fibroblasts, and the effect is better than that of commercially available recombinant type I collagen.

[0061] Example 5 Evaluation of the cell adhesion promoting activity of recombinant humanized type I collagen

[0062] The recombinant humanized type I collagen prepared in Example 2 was dissolved in PBS and added at a final concentration of 0.05 mg / ml and 0.10 mg / ml, respectively. 50 μL of the recombinant humanized type I collagen solution was added to each well of a 96-well cell culture plate and placed at 37°C for 2 h. PBS was added to the control wells. BALB / 3T3 cells were digested with trypsin, counted, and 5×10 4 Cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. PBS was washed three times to remove non-adherent cells, and then 200 μL DMEM medium was added; 10 μL CCK-8 reagent was added to each well, and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours before removal. The absorbance values ​​of the 96-well plate at 450nm and 630nm were read with an ELISA reader, and the absorbance was measured at 450nm with 630nm as the reference wavelength, and the measurement results were recorded.

[0063] Cell adhesion promotion rate = (450nm absorbance of the experimental group - 450nm absorbance of the negative control group) / 450nm absorbance of the negative control group × 100%.

[0064] In this example, the control group was a commercially available recombinant type I collagen sample solution, which was added into the cell culture medium at a concentration of 0.10 mg / ml, with 3 parallel samples in each group.

[0065] The experimental results are as follows Figure 4 As shown, the results show that at a concentration of 0.10 mg / ml, the cell adhesion promoting rates of the collagen in the control group and the recombinant humanized type I collagen of the present invention on BALB / c 3T3 cells are 44.5±3.5% and 42.5±6.6%, respectively. The cell adhesion promoting effects of the two are comparable, and both are superior to 24.5±3.4% of the recombinant humanized type I collagen of the present invention at a concentration of 0.05 mg / ml, indicating that the recombinant humanized type I collagen prepared by the present invention has good cell adhesion promoting activity.

[0066] Based on the above embodiments, the recombinant humanized type I collagen expressed by the present invention has uniform molecular weight, good solubility, and good cell adhesion and proliferation properties, and can be applied in various fields, such as cosmetics or pharmaceutical and medical products.

[0067] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the specification only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A recombinant humanized type I collagen, characterized in that: The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the recombinant humanized type I collagen according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pET-28a.

5. A recombinant bacterium expressing the recombinant humanized type I collagen according to claim 1, wherein the recombinant bacterium is Escherichia coli BL21 (DE3).

6. The method for producing recombinant humanized type I collagen according to claim 1, characterized in that: The method comprises the following steps: inoculating a recombinant bacterium expressing the recombinant humanized type I collagen described in claim 1 into a fermentation medium at an inoculation rate of 8-12%, fermenting and culturing for 30-40 hours, breaking the bacterial body and centrifuging to obtain a supernatant containing the recombinant humanized type I collagen described in claim 1, and purifying and desalting to obtain the recombinant humanized type I collagen described in claim 1.

7. A composition, characterized in that The composition comprises the recombinant humanized type I collagen according to claim 1, or the nucleic acid molecule according to claim 2, or the recombinant expression vector according to any one of claims 3-4, or the recombinant bacteria according to claim 5.

8. Use of the composition according to claim 7 in the preparation of cosmetics or medical device products.

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