Collagenase as well as coding gene and application thereof

Abstract

The invention relates to collagenase as well as a coding gene and application thereof. The collagenase provided by the invention is a protein (a) or (b), wherein (a) is a protein composed of an amino acid sequence as shown in SEQ ID No: 1; and (b) a protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a), has collagen hydrolysis or fat dissolving activity and is derived from (a). The collagenase provided by the invention can be applied to cell separation and tissue digestion, fat dissolution or collagen dissolution, and can also be applied to preparation of medicines for treating Doltt's contracture, Petroleum disease and lumbar disc herniation and degreasing in the field of beauty.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzyme engineering, and in particular to a collagenase, a coding gene thereof and an application thereof. BACKGROUND

[0002] Collagen is a natural protein widely present in mammals, accounting for more than 30% of the total protein content in the human body, and is the main component of connective tissue. It is widely present in skin, bone, cartilage, tendon, ligament, blood vessels and cornea, and is an important protein for maintaining tissue structure and function. Collagenase is a proteolytic enzyme that can specifically degrade collagen protein, and is widely present in bacteria, animal tissues and some parasites. Its main function is to degrade collagen protein in the extracellular matrix, thereby playing an important role in tissue remodeling, cell migration, tissue digestion and the like. At present, only a few collagenases are available on the market, such as collagenase derived from Clostridium histolyticum, which has commercial application, trademark More types of collagenase still need to be developed. SUMMARY

[0003] To solve the above technical problems, the present application provides a collagenase, a coding gene thereof and an application thereof. The collagenase provided by the present application is named HmCol, is derived from Hathewaya massiliensis, and is a protein with hydrolysis collagen or lipolysis activity.

[0004] In a first aspect, the present application provides a collagenase, which is a protein (a) or (b):

[0005] (a) a protein consisting of the amino acid sequence shown in SEQ ID No: 1;

[0006] (b) a protein derived from (a) with hydrolysis collagen or lipolysis activity by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a).

[0007] In some embodiments of the present application, the amino acid sequence of the protein (b) is shown in SEQ ID No: 2.

[0008] The amino acid sequence of the protein (b) shown in SEQ ID No: 2 in the present application is obtained by mutating E at position 452 to D from the amino acid sequence of the protein (a) shown in SEQ ID No: 1.

[0009] In a second aspect, the present application provides a coding gene of the collagenase according to the first aspect.

[0010] In some embodiments of the present application, the nucleotide sequence encoding the gene is a DNA molecule as shown in SEQ ID No: 3.

[0011] In the present application, SEQ ID No: 3 consists of 3003 bases, and the coding sequence is from the 1st to the 3003rd base at the 5' end.

[0012] In a third aspect, the present application provides a recombinant expression vector comprising the gene-encoding gene according to the second aspect.

[0013] The recombinant expression vector for constructing the gene-encoding gene of the collagenase described above is a plasmid expression vector in the field of genetic engineering, and feasible expression vectors include pET series vectors, pUC series vectors, pGEX series vectors, etc.

[0014] In a fourth aspect, the present application provides a transgenic cell line comprising the gene-encoding gene according to the second aspect.

[0015] In a fifth aspect, the present application provides a host bacterium comprising the gene-encoding gene according to the second aspect.

[0016] The host bacterium described in the present application includes E. coli BL21, E. coli Rosetta (DE3), etc.

[0017] In a sixth aspect, the present application provides the collagenase according to the first aspect for use in cell separation and tissue digestion, fat dissolution or collagen dissolution.

[0018] In the present application, the method for using collagenase for cell separation and tissue digestion is to add the recombinant collagenase into complex tissues (such as pancreas, liver, adipose tissue, heart, lung, skin, etc.) for separation of primary cells, and the tissue is dispersed into a single cell suspension by collagenase digestion, so as to perform flow cytometry, cell culture or other experiments.

[0019] In the present application, the method for using collagenase for fat dissolution or collagen dissolution is to inject the recombinant collagenase into subcutaneous fat for fat dissolution or collagen dissolution.

[0020] In a seventh aspect, the present application provides the collagenase according to the first aspect for use in the preparation of a medicament for treating Dupuytren's contracture, Peyronie's disease, and lumbar disc herniation.

[0021] In an eighth aspect, the present application provides the collagenase according to the first aspect for use in fat removal in the field of beauty.

[0022] The technical solution provided by the embodiments of the present application has the following advantages compared with the prior art.

[0023] The present application provides a new collagenase with collagen hydrolysis activity and its coding gene, which can be widely used in the field of biomedicine; the collagenase provided by the present application can be flexibly changed in activity through mutation, and can be used for lipolysis reaction in the field of beauty; the collagenase provided by the present application has high stability, and has the prospect of industrial application; the collagenase provided by the present application can be used for cleaning necrotic tissue in a wound and promoting tissue repair, and is applied to the preparation of a drug for treating Dupuytren contracture, Peyronie's disease, lumbar disc herniation and the like. BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings constituting a part of this specification illustrate embodiments consistent with the present application and, together with the specification, serve to explain principles of the present application.

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows, and obviously, other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0026] Figure 1 SDS-PAGE diagram of collagenase HmCol expressed in the embodiment 1 of the present application;

[0027] Figure 2 Liquid chromatogram of collagen protein analog catalyzed by collagenase HmCol in the embodiment 1 of the present application;

[0028] Figure 3 Optimal reaction temperature characterization diagram of collagenase HmCol and HmColM of the present application;

[0029] Figure 4 Optimal pH characterization diagram of collagenase HmCol and HmColM of the present application;

[0030] Figure 5 Lipolysis reaction characterization diagram of collagenase HmCol and HmColM of the present application;

[0031] Figure 6 Cell separation and tissue digestion characterization diagram of collagenase HmCol with different concentrations of the present application. DETAILED DESCRIPTION

[0032] In order to more clearly illustrate the above-mentioned purposes, features and advantages of the present application, the schemes of the present application will be further described below. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0033] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order to avoid obscuring the present application.

[0034] The reagents and instruments used in the examples are commercially available unless otherwise specified.

[0035] In Examples 1 and 2, the basic genetic engineering methods used are described in the Molecular Cloning: A Laboratory Manual, Third Edition (J. Sambrook et al., Science Press, 2002).

[0036] Example 1

[0037] This example provides a collagenase HmCol, and a preparation method thereof is as follows:

[0038] (1) Obtaining of collagenase HmCol

[0039] The original gene sequence of collagenase HmCol derived from Hathewaya massiliensis (Gene ID: WP_185903632) was obtained from Genbank. The original gene was codon-optimized to be suitable for heterologous expression in Escherichia coli and the artificial predicted N-terminal signal peptide was deleted (the protein without the signal peptide is not active), and a histidine tag was added at the C-terminal of the sequence for protein purification, to obtain the nucleotide sequence shown in SEQ ID No: 3 in the sequence listing. The nucleotide sequence shown in SEQ ID No: 3 was directly synthesized by solid-phase phosphoramidite chemistry, and was directly connected to the pET30a(+) vector by NdeI and XhoI sites by Gibson Assembly gene recombination;

[0040] (2) Expression and purification of recombinant protein

[0041] The above constructed plasmid pET30a(+)-HmCol was introduced into Escherichia coli BL21 (DE3) by heat shock method to obtain transformant E. coli BL-21 (pET-30-HmCol); 10 μL of the transformant was inoculated into LB liquid medium (containing kanamycin) and cultured at 37°C for 3 hours, 60 μL of 500 mol / L IPTG was added, and the expression was induced at 16°C overnight to obtain an induced expression mixture. The induced expression mixture was centrifuged and the bacterial cells were collected, which were the recombinant Escherichia coli cells; the bacterial cells were suspended in phosphate buffer and were ultrasonically broken. The ultrasonically broken bacterial solution was centrifuged (12000 rpm), and the supernatant was collected, which was the crude extract of the collagenase.

[0042] (3) The supernatant is purified by a Ni-NTA nickel column (Ni-NTA His-Tag Purification Agarose) and a molecular sieve, so that the collagenase is obtained, and the purification effect of the collagenase is detected by SDS-PAGE.

[0043] The detection results are shown in Figure 1 , Figure 1 The SDS-PAGE diagram of the collagenase HmCol expression is shown in Figure 1 It can be seen that the collagenase prepared in this embodiment has a good purification effect, and most of the impurities are removed.

[0044] In this embodiment, the amino acid sequence of the HmCol protein is shown in SEQ ID No: 1.

[0045] MVQEESRNYTLADLRRLSYYDLVDLLVKTDVYNLTDLFQYSEDARQFYGDKTRMSFLMDEIQRRGSQYTDIDSKGIPTLIEVVRAGFYQGFYHKELEELNKREFRERAIPAVLSIQRNPNFVLGTEVQDKIVSSTGLFVWNGASNPEVINNFTPILKQYTKNMNIFVNDNAKGTALFNVLGGPSYDISTYISTTKKSPEETQWYRKIDGFINEVKNIAFIGEVNEKNSWIIDNGIYHIASLGKLHSNSKIGIETLTQTMKMYPYLSQQHLQSAEQIRRNYNGIDAAGNKIDMEKLKEEGKAKYCPKTYTFDNGKVIIKAGDRVEEEKIKRLYWASKEVNSQFFRVMGRDNPLEAGNPDEILTMVIYNSPDEYTLNSVLYGYETNNGGMYIEGKGTFFTYERTPEESTYTLEELFRHEYTHYLQGRYAVPGQWGDTELYKNDRLTWYEEGGAELFAGSTRTSGVLPRKSIINNIRTTTRNNRYKVSDTVNSKYASGFEFYNYACVFVDYMYNKDITTLDKINNIVKSNDVKAYDDYMKELSSNYSLNDRYQDHMQKLIDDYDNLTVPLVSDDYLIRHAYKNPNEIYSEITNVAKLKDVKTEINKSQFFKTFTLRGKYSAGTSKGKIEDYKQMDKLTNEFLKKLDAYSWSGYKTVTAYFTNYKVDASNNVTYDVAFHGYMPDENDSTNSLPYGKINGPYNGTQNTKIKFSSEGSFDPDGKIVSYEWDFGDGNKSKDENPEHSYAKVGTYNVTLKVTDDKGGYSISKTTVGVKDLSENQLPVVYMEVPTTAYVDKEVTFYGKGTYDPDGSVAGYQWDFGDGSDVSYVQNPTHLYRKKGEYTVTLKVMDNSGQMTEKTKKIVVTDPVYPINRETEPNDSKEMANGPILPGVSVNGTLDNTDYSDFFYFDVITPGEVKIDIAKLGYGGATWVVYDENNNAVAYATEDGYNLKGSFKAQKSGKYYIHLYMYSGQYMPYRINVSGSVGRGSENLYFQGALEHHHHHH

[0046] In this embodiment, the nucleotide sequence of the gene is shown as SEQ ID No: 3:

[0047]

[0048] Example 2

[0049] The present example provides a collagenase HmColM, the preparation method of which is different from the preparation method of Example 1 in that the collagenase HmCol is mutated by solid-phase phosphoramidite chemistry to mutate the coding GAA at positions 1354-1356 of the nucleotide sequence shown in SEQ ID No: 3 to GAT, and the amino acid sequence corresponding mutation is to mutate E at position 452 to D.

[0050] In the present example, the amino acid sequence of the HmColM protein (E452D mutant) is shown in SEQ ID No: 2:

[0051] MVQEESRNYTLADLRRLSYYDLVDLLVKTDVYNLTDLFQYSEDARQFYGDKTRMSFLMDEIQRRGSQYTDIDSKGIPTLIEVVRAGFYQGFYHKELEELNKREFRERAIPAVLSIQRNPNFVLGTEVQDKIVSSTGLFVWNGASNPEVINNFTPILKQYTKNMNIFVNDNAKGTALFNVLGGPSYDISTYISTTKKSPEETQWYRKIDGFINEVKNIAFIGEVNEKNSWIIDNGIYHIASLGKLHSNSKIGIETLTQTMKMYPYLSQQHLQSAEQIRRNYNGIDAAGNKIDMEKLKEEGKAKYCPKTYTFDNGKVIIKAGDRVEEEKIKRLYWASKEVNSQFFRVMGRDNPLEAGNPDEILTMVIYNSPDEYTLNSVLYGYETNNGGMYIEGKGTFFTYERTPEESTYTLEELFRHEYTHYLQGRYAVPGQWGDTELYKNDRLTWYEEGGADLFAGSTRTSGVLPRKSIINNIRTTTRNNRYKVSDTVNSKYASGFEFYNYACVFVDYMYNKDITTLDKINNIVKSNDVKAYDDYMKELSSNYSLNDRYQDHMQKLIDDYDNLTVPLVSDDYLIRHAYKNPNEIYSEITNVAKLKDVKTEINKSQFFKTFTLRGKYSAGTSKGKIEDYKQMDKLTNEFLKKLDAYSWSGYKTVTAYFTNYKVDASNNVTYDVAFHGYMPDENDSTNSLPYGKINGPYNGTQNTKIKFSSEGSFDPDGKIVSYEWDFGDGNKSKDENPEHSYAKVGTYNVTLKVTDDKGGYSISKTTVGVKDLSENQLPVVYMEVPTTAYVDKEVTFYGKGTYDPDGSVAGYQWDFGDGSDVSYVQNPTHLYRKKGEYTVTLKVMDNSGQMTEKTKKIVVTDPVYPINRETEPNDSKEMANGPILPGVSVNGTLDNTDYSDFFYFDVITPGEVKIDIAKLGYGGATWVVYDENNNAVAYATEDGYNLKGSFKAQKSGKYYIHLYMYSGQYMPYRINVSGSVGRGSENLYFQGALEHHHHHH

[0052] Example 3

[0053] Determination of the properties of collagenase:

[0054] 1. Analysis of collagenase hydrolysis activity

[0055] The collagenase HmCol prepared in Example 1 of this invention was subjected to liquid chromatography analysis of the collagen-like peptide substrate (because collagen molecules are too large to be directly measured by liquid chromatography, collagen-like peptides were used for indirect measurement).

[0056] The synthetic peptide 4-benzyloxycarbonyl(PZ)-Pro-Leu-Gly-Pro-D-Arg was used as a substrate, and Ca2+ was added. + To optimize the enzymatic reaction, a typical reaction system consisted of 3 μL of 12.3 mM substrate, 10 μL of 0.2 M CaCl2, 46 μL of 0.1 M Tris buffer (pH 7.1), and 1 μL of 1 mg / mL collagenase solution, reacted at 25 °C for 30 min. HPLC analysis was performed on a Shimadzu LC-20A system equipped with an SPD-16 detector, an LC-16P pump, and a SilGreen column (5 μm, 12 nm, 4.6 × 150 mm). The mobile phase was water (A) and acetonitrile (B) containing 0.1% trifluoroacetic acid, with a gradient elution program of: 0–2 min 20% B, 2–10 min to 50% B, 10–20 min to 95% B, 20–25 min to 95% B, and 25–27 min back to 20% B. The flow rate was 1 mL / min, the column temperature was 40℃, the detection wavelength was 320 nm, and the injection volume was 20 μL. Results are as follows: Figure 2 As shown:

[0057] Figure 2 This is a chromatogram of the liquid phase analysis of collagenase HmCol catalyzing collagen-like peptide substrates. As shown in the figure, in the control group, the substrate peak appears at 17 minutes; after incubation with HmCol, the substrate peak weakens, while a new product peak appears. Figure 2 It can be seen that the collagenase prepared in this embodiment has good hydrolytic activity against collagenase polypeptide analogs.

[0058] 2. Optimal Temperature Analysis

[0059] The collagenases HmCol and HmColM purified in Examples 1 and 2 of this invention were subjected to enzymatic reactions under different temperature conditions to determine their optimal reaction temperature, with pH 7.1. The results are as follows: Figure 3 As shown:

[0060] Depend on Figure 3It can be seen that the collagenase HmCol obtained after purification has an optimal reaction temperature of 45 DEG C at pH 7.1, and the enzyme activity is nearly 100%, and the enzyme activity is still more than 75% at 55 DEG C; and the collagenase HmColM has an optimal reaction temperature of 45 DEG C at pH 7.1, the enzyme activity is 52%, and the enzyme activity is less than 20% at 55 DEG C.

[0061] In summary, the collagenase HmCol provided by the embodiments of the present application has high protein hydrolysis activity at 45-55 DEG C, and the hydrolysis activity of the collagenase HmColM is nearly 50% weaker than that of the collagenase HmCol, which means that the hydrolysis reaction of HmColM is more moderate.

[0062] 3. Optimal pH analysis

[0063] The collagenases HmCol and HmColM obtained by purifying the embodiments 1 and 2 were subjected to enzymatic reaction under different pH conditions to determine the optimal pH value. The buffer used was (Tris buffer), and the temperature was 25 DEG C. The results are shown in Figure 4 (A and B) as follows:

[0064] As shown in Figure 4 A, under the condition of 25 DEG C, the optimal pH of the collagenase HmCol is 7.5, and the enzyme can maintain a high enzyme activity in the pH range of 7.0-8.0; and as shown in Figure 4 B, under the condition of 25 DEG C, the optimal pH of the collagenase HmColM is 7.0, and the enzyme can maintain a high enzyme activity in the pH range of 7.0-8.0.

[0065] Application example 1

[0066] The collagenases described in embodiments 1 and 2 were respectively applied to the dissolution of fat:

[0067] Freshly slaughtered pig back fat-rich tissue meat was injected with a control group (PBS buffer), collagenase HmCol obtained in embodiment 1 and collagenase HmColM obtained in embodiment 2, respectively, and the fat tissue dissolution was observed after 5 hours, and the results are shown in Figure 5 (A, B, C) as follows:

[0068] As shown in Figure 5 (A, B, C), under the same time conditions, compared with the control group, the collagenase HmCol can make the fat cells apoptotic and liquefied after injection; the collagenase HmColM can make the fat cells apoptotic and liquefied after injection, but the degree of apoptosis and liquefaction is lower than that of the collagenase HmCol, which indicates that the collagenase HmColM after mutation is more moderate in the reaction of fat removal in the medical and cosmetic fields.

[0069] Application Example 2

[0070] Different concentrations of the collagenase HmCol described in Example 1 were applied to the digestion and cell separation of pancreatic tissue:

[0071] Collagenase HmCol at concentrations of 0.1 mg / mL, 0.3 mg / mL, 0.6 mg / mL, and 1.0 mg / mL, along with a control group (PBS buffer), were added to minced pancreatic tissue. The tissue was incubated at 37°C for 5 hours to isolate islet cells. The pancreatic tissue was then dispersed into a single-cell suspension by collagenase digestion. Results are as follows: Figure 6 As shown:

[0072] Depend on Figure 6 It is known that collagenase HmCol can efficiently hydrolyze the intercellular tissue, thereby achieving the effect of dispersing cells.

[0073] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0074] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. A collagenase, characterized in that, The collagenase is a protein (a) or a protein (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID No: 1; (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has hydrolytic collagen or lipolytic activity.

2. The collagenase according to claim 1, characterized in that, The amino acid sequence of the protein (b) is shown in SEQ ID No:

2.

3. A gene encoding collagenase as described in claim 1.

4. The encoding gene as described in claim 3, characterized in that, The nucleotide sequence of the encoding gene is the DNA molecule shown in SEQ ID No:

3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene as described in claim 3 or 4.

6. A transgenic cell line, characterized in that, The transgenic cell line contains the encoding gene as described in claim 3 or 4.

7. A host bacterium, characterized in that, The host bacterium contains the coding gene as described in claim 3 or 4.

8. The use of the collagenase as described in claim 1 in cell separation and tissue digestion, lipolysis, or collagen dissolution.

9. The use of the collagenase as described in claim 1 in the preparation of a medicament for treating Dupilot contracture, Peronni's disease, and lumbar disc herniation.

10. The application of collagenase as described in claim 1 in the field of cosmetic fat removal.

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