WO2023138668A1

WO2023138668A1 - Stable macromolecular type i recombinant collagen and use thereof

Info

Publication number: WO2023138668A1
Application number: PCT/CN2023/073217
Authority: WO
Inventors: 范代娣; 宇文伟刚; 贺婧; 段志广; 徐茹; 严建亚; 刘琳
Original assignee: 陕西巨子生物技术有限公司
Priority date: 2022-01-24
Filing date: 2023-01-19
Publication date: 2023-07-27
Also published as: CN114409807B; CN114409807A

Abstract

The present invention relates to a stable macromolecular type I recombinant collagen and a use thereof. The macromolecular type I recombinant collagen of the present invention is formed by performing multiple repetitions by using a short amino acid sequence derived from natural human type I collagen as a repeating unit, the short amino acid sequence being GAPGAPGSQGAPGLQ, and the number of repetitions being 75-110 times. The macromolecular type I recombinant collagen of the present invention has extremely good stability in an aqueous solution and can be stored in an aqueous solution for a long period of time.

Description

A kind of stable macromolecule type I recombinant collagen and its application

technical field

The invention belongs to the field of biotechnology, and relates to a novel genetic engineering recombinant collagen and its application.

Background technique

Collagen is a biological polymer protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation, and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.

The source of natural human collagen is limited. The natural collagen currently used in industry is mainly extracted from animal skin or bone by acid, alkali or enzymatic methods, and its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases, and at the same time, large-scale preparations have caused huge pressure on animal feeding on the supply side.

With the widespread application of genetic engineering technology, the bottleneck problem of large-scale production of human collagen has been successfully solved by using suitable engineering strains (Escherichia coli, Pichia pastoris, etc.) to express human collagen exogenously. However, when engineering strains such as Pichia pastoris genetically engineered strains are used to express human collagen, human collagen will be degraded during fermentation, purification and storage, which increases production costs and affects the performance of human collagen produced by this method.

It is speculated that the reason for the degradation of human collagen may be that its amino acid sequence contains more sites that are prone to hydrolysis. Therefore, technicians construct recombinant collagen by selecting and repeating short amino acid sequences from natural human collagen in order to avoid sites prone to hydrolysis, thereby improving the stability of collagen while maintaining the excellent properties of natural human collagen. However, the amino acid composition and distribution of the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen is relatively monotonous. Theoretically speaking, this will cause a large charge load on its surface, and it is difficult to reach a stable equilibrium state as a whole. Therefore, it is easy to hydrolyze and denature in aqueous solution, and there is a tendency that the shorter the short amino acid sequence repeat unit and the more repetitions, the more unstable the recombinant collagen molecule is in aqueous solution.

One can attempt to obtain recombinant collagens that are more resistant to degradation by mutating short amino acid sequences derived from native collagen as repeating units. However, the homology between the recombinant collagen obtained by such mutation and natural human collagen is reduced, and immunogenicity problems may arise, so it is not suitable for use in biomaterials that need to be in long-term contact with the human body.

On the other hand, tissue engineering materials such as dermal fillers are an important application direction of human collagen. As tissue engineering materials, collagen is required to have good mechanical strength and stability in aqueous solution (it can be stored in aqueous solution for a long time). In general, the higher the molecular weight of collagen, the better its mechanical strength and the lower its stability in aqueous solution, especially for recombinant collagen constructed by repeating short amino acid sequences from natural human collagen.

The molecular weight of each chain of natural human collagen is about 110-130kD. From a practical point of view, technical personnel generally believe that the molecular weight of collagen suitable for replacing natural human collagen as a tissue engineering material needs to reach more than 100kD. However, as mentioned above, the source of natural human collagen is limited, animal-derived collagen has the risk of spreading diseases, and human collagen expressed by genetic engineering is prone to degradation during fermentation, purification and storage. Recombinant collagen constructed by repeating acid sequences is unstable in aqueous solution, and recombinant collagen obtained through mutation engineering has immunogenicity problems. Therefore, how to obtain collagen suitable for replacing natural human collagen as tissue engineering material has become a limiting problem in this technical field.

Contents of the invention

In order to solve the above-mentioned technical problems existing in the prior art, the inventors conducted in-depth research. The inventors first conducted technical literature research on recombinant collagens constructed repeatedly from short amino acid sequences from natural human collagen, selected some short amino acid sequences from natural human type I collagen (the most widely used tissue engineering material) in the prior art, and then used these short amino acid sequences as repeating units to construct recombinant collagens with different molecular weights, and investigated the long-term storage stability of these recombinant collagens in aqueous solution, in order to obtain large molecular weight (above 100kD) recombinant collagens that can be stored in aqueous solution for a long time and can meet the mechanical strength requirements of tissue engineering materials .

As a result of the above research, the inventor unexpectedly discovered that the recombinant type I collagen obtained by repeating a section of pentadecadecanoid (G A P G A P G S Q G A P GL Q) from natural human type I collagen for 75 to 110 times has exceptionally excellent stability. It is specifically reflected in: (1) Although the length of its repeating unit is the shortest among all the recombinant collagens tested by the inventor, its stability in aqueous solution is the best; and it is generally believed that the shorter the repeating unit, the more monotonous the composition and distribution of amino acids, the greater the surface charge load of the recombinant collagen thus constructed, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze. (2) It is even more stable than the recombinant type I collagen obtained by repeating the 15-peptide 52 times, 62 times or 72 times, and it is generally believed that the more the number of repetitions, the greater the molecular weight, the greater the surface charge load of the recombinant collagen, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze.

Based on the above findings, the inventors have accomplished the present invention. That is, the present invention includes:

1. A macromolecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is as shown in SEQ ID No.: 1 (G A P G A P G S Q G A P GL Q), and the number of repetitions is 75 to 110 times.

2. The macromolecular type I recombinant collagen according to item 1, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

3. The macromolecular type I recombinant collagen according to item 1, which has a molecular weight of more than 120kD.

4. The macromolecular type I recombinant collagen according to item 1, further bearing a tag for easy purification.

5. The macromolecular type I recombinant collagen according to item 4, wherein the tag is a His tag, a Flag tag or a c-Myc tag.

6. Use of the macromolecular type I recombinant collagen according to any one of items 1 to 5 as a tissue engineering material.

7. The use according to item 6, wherein the tissue engineering material is selected from the group consisting of dermal fillers, artificial bones, artificial skin, oral cavity absorbable biofilms, bone implants, vascular scaffolds, intercellular matrix scaffolds and collagen sponges.

8. Use of the macromolecular type I recombinant collagen according to any one of items 1 to 5 as a dermal filler, artificial bone, artificial skin, oral cavity absorbable biofilm, bone implant, vascular scaffold, intercellular matrix scaffold or collagen sponge.

9. An aqueous collagen solution, comprising the macromolecular type I recombinant collagen described in any one of items 1-5.

10. The collagen aqueous solution according to item 9, which has been stored at room temperature for more than 3 months, preferably for more than 6 months, more preferably for more than 12 months; or has been stored at 4°C for more than 12 months, preferably for more than 24 months, more preferably for more than 36 months.

The inventors are conducting more in-depth research on the reason why the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability. Preliminary findings suggest that this may be due to:

The stability of recombinant collagen that repeats with a certain amino acid sequence as a repeat segment is closely related to the surface charge, and the surface charge is related to the composition of amino acids and the spatial structure of the protein. After reaching a certain number of repetitions, a certain spatial structure is just formed, making the surface load in a balanced or near-balanced state, so it will show an abnormally stable state. The inventor just found that the 15 amino acid repeat sequence collagen is in the range of load balance, so the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability.

Description of drawings

FIG. 1 is an SDS-PAGE electrophoresis image of a portion of type I recombinant collagen prepared in Example 1. Take No.4, 6, 7, and 10 as examples for control proteins.

Fig. 2 is the HPLC picture of part of the test samples prepared in Example 2 after standing for 12 months. Take No.4, 7, 10, and 13 as examples for control proteins.

Fig. 3 is the infrared spectrogram of No.1 and No.6 type I recombinant collagen.

Fig. 4 is the Raman spectrum of No.1 and No.6 type I recombinant collagens.

Detailed ways

The present invention will be described in detail through specific examples below. It should be pointed out that these descriptions are only exemplary descriptions, and are not intended to limit the scope of the present invention.

General description: All the enzymes used in the specific embodiment were purchased from TaKaRa Company, the plasmid DNA extraction kit and the DNA gel recovery kit were purchased from Beijing Suolaibao Company, and the gene reorganization kits (Reorganization Kits) were purchased from Tiangen Biology, and the specific operations were carried out in accordance with the instructions of the kits.

Example 1. Preparation of Various Type I Recombinant Collagens Using Yeast Expression System

1. Construction of yeast expression strains

Yeast expression strains expressing No. 1-18 type I recombinant collagen shown in Table 1 were constructed. The specific operation is: after optimizing the codon preference of Pichia pastoris, synthesize the corresponding target gene by whole gene synthesis, add SnaB I and Not I restriction sites at both ends of the gene respectively, perform double digestion with SnaBI and Not I enzymes on the target gene, and connect it with pPIC9k, which has also been digested by SnaB I and Not I enzymes, under the action of T4 ligase. Transformants, linearized with SacI after plasmid extraction Afterwards, electroporation was transferred into Pichia pastoris GS115 competent cells, and multi-copy transformants were screened with G418 resistance plate, which was the expression strain of type I recombinant collagen.

Table 1 Type I recombinant collagen expressed by each yeast expression strain

2. Induced expression of target protein

(1) Pick a single colony of the yeast expression strain and add it to 5ml YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and cultivate overnight at 30°C at 200rpm for activation;

(2) Inoculate 100 ml of BMGY liquid medium with 1% inoculum size, culture at 30°C and 200 rpm until OD ₆₀₀ =6.0-9.0;

(3) Under the action of 1500g centrifugal force, centrifuge at 25°C for 6 minutes to collect the bacterial cells, suspend them in 200ml BMMY liquid medium, make the initial concentration OD ₆₀₀ =1.0, and cultivate them at 30°C and 200rpm;

(4) Methanol was added every 24 hours, with a final concentration of 0.5-1.0%, to induce expression;

(5) After induction for 72 hours, the culture solution was taken and centrifuged at 12,000 rpm for 2 minutes, and the supernatant was taken.

3. Preparation of type I recombinant collagen

After the supernatant prepared by fermentation is concentrated by ultrafiltration with a 30kD ultrafiltration membrane, the CM ion exchange column is used for column separation, eluted with 35% NaCl solution, the eluate is collected, desalted and concentrated, and then freeze-dried to prepare type I recombinant collagen. Take 0.1g of lyophilized powder and dissolve it in 100ml of normal saline, fully dissolve it and perform SDS-PAGE gel electrophoresis to confirm the molecular weight and protein electrophoresis purity.

The results showed that all the 18 expression strains constructed could successfully express the target protein, and the electrophoresis purity of the prepared protein after separation and purification was all above 99%. The electrophoresis results are shown in Figure 1 .

Embodiment 2: Stability experiment of various type I recombinant collagens in aqueous solution

AExperimental material

The materials used in the experiment were type I recombinant collagen No.1--18 prepared in Example 1.

BExperimental method

Prepare the experimental materials in A with ddH ₂ O to form a protein solution with a protein concentration of 1 mg/mL, filter it with a 0.22 μm sterile filter in an ultra-clean workbench, divide it into sterile centrifuge tubes, seal it, and place it at 25°C±2°C. Samples were taken at 0 months, 6 months, and 12 months, and 3 tubes were sampled each time.

C Experimental results

The test results are as follows:

Table 2 Reconstituted collagen solution 12 months stability test result (purity, %)

It is generally believed that (1) for recombinant collagen with the same or similar molecular weight, the shorter the repeating unit, the more monotonous the composition and distribution of amino acids, the greater the surface charge load, the less likely it is to reach a stable equilibrium state, and thus easier to hydrolyze; The greater the sub-weight, the greater the surface charge load of the recombinant collagen, and the less likely it is to reach a stable equilibrium state, so it is more likely to be hydrolyzed.

However, as can be seen from Table 2, the type I recombinant collagen (No. 1-3) of the present invention exhibits exceptionally excellent stability in aqueous solution, and its purity can still be as high as 97% or more after being placed in aqueous solution for 12 months (see Figure 2A-G).

Example 3: Preliminary research on the reasons why type I recombinant collagen No. 1-3 has abnormal stability in aqueous solution

1) Infrared Spectroscopy Determination

Reconstituted collagens No.1 and No.6 in Table 1 were formulated into solutions, and infrared spectroscopy was carried out. Bruker OPUS7.2 was used to perform Fourier deconvolution on the spectral data, and the spectral data in the 1700-1600 cm- ¹ band was intercepted. Second-order derivative peak fitting was performed with peakfit v4.12. The processed data was plotted with orgin to obtain the secondary structure distribution, and the relative content of the secondary structure was calculated. The comparison results of the secondary structures of the two proteins are shown in Table 3 and Figure 3.

The secondary structure of No.1 and No.6 recombinant collagen of table 3 infrared spectrum determination

2) Raman Spectroscopy Determination

Recombinant collagens No.1 and No.6 in Table 1 were formulated into solutions, and Raman spectra were measured. ThermoFisher Omnic9.2 was used to smooth the baseline of the spectral data, and the spectral data in the 1700-1600 cm- ¹ band was intercepted, and the second-order derivative peak fitting was performed with peakfit v4.12. The processed data was plotted with orgin to obtain the secondary structure distribution, and the relative content of the secondary structure was calculated. The comparison results of the secondary structures of the two proteins are shown in Table 4 and Figure 4.

The secondary structure of No.1 and No.6 recombinant collagen determined by Raman spectroscopy in table 4

It can be seen from the measurement results of Example 3 that the recombinant collagens with the same repeating unit but different repeating numbers have large differences in secondary structures, which also implies that there are differences in the tertiary structures of the two. It can be speculated that the spatial structure of Type I recombinant collagen No. 1-3 makes the surface charge more balanced, thus showing good stability in aqueous solution.

Claims

A macromolecular type I recombinant collagen, which is composed of a short amino acid sequence derived from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is shown in SEQ ID No.: 1 (G A P G A P G S Q G A P G L Q), and the number of repetitions is 75 to 110 times.
The macromolecular type I recombinant collagen according to claim 1, wherein the number of repetitions is 80-105 times, preferably 82-102 times.
The macromolecular type I recombinant collagen according to claim 1, which has a molecular weight of more than 100kD.
The macromolecule type I recombinant collagen according to claim 1, which also has a label that makes it easy to purify.
The macromolecular type I recombinant collagen according to claim 4, wherein the tag is a His tag, a Flag tag or a c-Myc tag.
Use of the macromolecular type I recombinant collagen according to any one of claims 1 to 5 as a tissue engineering material.
The use according to claim 6, wherein the tissue engineering material is selected from the group consisting of dermal fillers, artificial bones, artificial skin, oral cavity absorbable biofilms, bone implants, vascular scaffolds, intercellular matrix scaffolds and collagen sponges.
Use of the macromolecular type I recombinant collagen according to any one of claims 1 to 5 as a dermal filler, artificial bone, artificial skin, oral cavity absorbable biofilm, bone implant, vascular scaffold, intercellular matrix scaffold or collagen sponge.
An aqueous collagen solution comprising the macromolecular type I recombinant collagen according to any one of claims 1-5.
The aqueous collagen solution according to claim 9, which has been stored at room temperature for more than 3 months, preferably for more than 6 months, more preferably for more than 12 months; or has been stored at 4°C for more than 12 months, preferably for more than 24 months, more preferably for more than 36 months.