Recombinant collagen and its expression method in Pichia pastoris

Abstract

The present application relates to the field of biotechnology, and specifically discloses a recombinant collagen protein and a method for expressing it in Pichia pastoris. The amino acid sequence of the basic structural unit of the recombinant collagen protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the basic structural unit is shown in SEQ ID NO: 2. The recombinant collagen protein Col (I+III) of the present application contains specific basic structural units, which can increase the expression level of collagen protein in host cells, as well as improve the stability and activity of collagen protein, thereby significantly promoting the proliferation of fibroblasts; and, the preparation of the collagen protein of the present application by genetic engineering can better increase the expression level of recombinant collagen protein in host cells and promote the proliferation of fibroblasts.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant collagen and a method for expressing the same in Pichia pastoris. Background Art

[0002] Collagen has good biocompatibility, low immunogenicity, biodegradability and good mechanical properties. Currently, collagen is widely used in biomedicine and biomedical materials. Collagen-based biomaterials have been used in skin tissue repair, soft tissue filling materials, bone tissue regeneration fillers, surgical hemostatic materials, tissue filling materials, artificial blood vessels, etc.

[0003] Collagen comes primarily from two sources: collagen extracted from animal tissue and recombinant collagen produced using recombinant DNA technology. Collagen extracted from animal tissue may retain a complete collagen molecular structure, but it also carries the risk of viral introduction and exhibits some immunogenicity. Collagen produced using recombinant DNA technology offers improved biocompatibility, water solubility, and stability, and eliminates the risk of viral introduction.

[0004] The U.S. Food and Drug Administration (FDA) has designated Pichia pastoris as a generally recognized as safe (GRAS) microorganism, making it widely used in medicine and food. The Pichia pastoris expression system offers advantages such as minimal secretion of contaminants, low culture costs, and mature fermentation processes. Numerous exogenous proteins have been successfully expressed in this system, including epidermal growth factor (EGF), human serum albumin (HSA), hepatitis B surface antigen (HBsAg), vaccines, insulin products, and enzyme preparations. Furthermore, Pichia pastoris is widely used as a host cell for the food industry and biomaterial expression. For example, cellulases, β-mannanases, and hemicellulases can be efficiently expressed in Pichia pastoris.

[0005] Due to the specific and repetitive amino acid composition of collagen, how to improve the stability and expression level of collagen and whether it can form the correct folding structure in the process of preparing collagen by recombinant DNA technology are key aspects restricting its application. Summary of the Invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a recombinant collagen and a method for expressing the same in Pichia pastoris.

[0007] To achieve the above objectives, the technical solutions adopted in this application are:

[0008] The present application provides a recombinant collagen protein, wherein the amino acid sequence of the basic structural unit of the recombinant collagen protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the basic structural unit is shown in SEQ ID NO: 2.

[0009] After extensive research and experiments, the inventors of the present application discovered that the recombinant collagen (named Col(I+III)) composed of the above basic structural units can significantly promote the proliferation of fibroblasts.

[0010] In some specific embodiments, recombinant collagen Col (I+III) can promote the proliferation of fibroblasts. At a concentration of 80 μg / mL, the recombinant collagen Col (I+III) can promote the maximum proliferation rate of fibroblasts to 41.3%.

[0011] The present application also provides a DNA sequence encoding the recombinant collagen, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0012] The present application provides an expression vector comprising a DNA sequence encoding the recombinant collagen.

[0013] As a preferred embodiment of the expression vector described in the present application, the expression vector includes the pPICZaA plasmid.

[0014] The expression vector of the present application may also contain plasmids commonly used in the art, and is not limited to the pPICZaA plasmid.

[0015] The present application provides a genetically engineered bacterium, which includes the expression vector.

[0016] As a preferred embodiment of the genetically engineered bacteria described in the present application, the genetically engineered bacteria include Pichia pastoris. Genetically engineered bacteria are not limited to Pichia pastoris, but also include common genetically engineered bacteria in the art.

[0017] The present application also provides a method for expressing the above-mentioned recombinant collagen in Pichia pastoris, comprising the following steps:

[0018] S1. The DNA sequence encoding the recombinant collagen and the Pichia pastoris expression vector pPICZaA are linearized by restriction endonuclease digestion, the linearized fragments are transformed into the competent Pichia pastoris host strain GS115, and cultured, a single colony is selected for culture, the fermentation supernatant is collected and then subjected to agarose gel electrophoresis to select the strain expressing the recombinant collagen;

[0019] S2, inoculating the strain obtained in step S1 into a liquid culture medium to obtain a fermentation seed solution; inoculating the fermentation seed solution into a fermentation tank for cultivation, adding glycerol, controlling the dissolved oxygen to maintain DO at 20-30%, until the bacterial OD 600 At 200-250, stop adding glycerol;

[0020] S3. After stopping the flow addition of glycerol, methanol flow addition was started for induction, and the dissolved oxygen was controlled to maintain DO at 20-30%, and the fermentation liquid was obtained, and then subjected to agarose gel electrophoresis for separation and purification.

[0021] Collagen is produced through genetic engineering, which requires the selection of appropriate collagen domains. Rational optimization of the amino acid sequence of the selected collagen domains can increase the expression level of collagen in host cells, as well as improve the stability and activity of collagen.

[0022] As a preferred embodiment of the method for expressing recombinant collagen in Pichia pastoris described in the present application, in step S1, if agarose gel electrophoresis shows a clear electrophoretic band at the 35 kDa position, it means that the recombinant collagen described in claim 1 has been expressed.

[0023] As a preferred embodiment of the method for expressing recombinant collagen in Pichia pastoris described in the present application, the selecting of a single colony for cultivation comprises the following steps:

[0024] Select a single colony and inoculate it into YPD liquid medium and culture it at 30℃ and 220rpm. Then transfer it to BMGY liquid medium and culture it at 30℃ and 220rpm for 24h. Then centrifuge it at 5000rpm for 10 minutes, pour out the supernatant to collect the bacteria, and finally replace the BMGY liquid medium with BMMY liquid medium. Then add inducer and induce it at 30℃ and 220rpm, and take the fermentation supernatant.

[0025] As a preferred embodiment of the method for expressing recombinant collagen in Pichia pastoris described in the present application, in step S3, the methanol induction time is 24 to 72 hours. Preferably, the methanol induction time is 72 hours.

[0026] Different induction times of methanol have a great influence on the expression of recombinant collagen. The expression concentrations of recombinant collagen corresponding to 24h, 48h and 72h induction are 0.47mg / ml, 1.2mg / ml and 2mg / ml, respectively. This indicates that the expression level of recombinant collagen in Pichia pastoris GS115 is the highest when the methanol induction time is 72h.

[0027] As a preferred embodiment of the method for expressing recombinant collagen in Pichia pastoris described in the present application, in step S2, the liquid culture medium includes YPD liquid culture medium and BMGY liquid culture medium.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] The present application provides a recombinant collagen protein and a method for expressing it in Pichia pastoris. The recombinant collagen protein Col(I+III) of the present application contains specific basic structural units, which can increase the expression level of collagen protein in host cells, as well as improve the stability and activity of collagen protein, thereby significantly promoting the proliferation of fibroblasts; and, the preparation of the collagen protein of the present application by genetic engineering can better increase the expression level of recombinant collagen protein in host cells and promote the proliferation of fibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Electrophoresis diagram of nucleic acid extracted from the expression vector pPICZaA-Col(I+III) plasmid (1: non-linearized pPICZaA-Col(I+III) plasmid, 2: SacI-linearized pPICZaA-Col(I+III) plasmid);

[0031] Figure 2 Electrophoresis results of electroporation of pPICZaA-Col(I+III) plasmid into competent cells of Pichia pastoris GS115 host strain or induced expression of single clones of Pichia pastoris GS115 host strain (1: untransfected Pichia pastoris host strain GS115, 2-9: induced expression of different single clones selected after electroporation of pPICZaA-Col(I+III) plasmid into competent cells of Pichia pastoris GS115 host strain);

[0032] Figure 3 Figure 1 shows the electrophoresis results of high-density fermentation-induced expression of genetically engineered bacteria GS115-Col(I+III) (1: 5-fold dilution of the supernatant of the fermentation broth of the uninduced genetically engineered bacteria GS115-Col(I+III); 2-4: protein electrophoresis results of the supernatant of the fermentation broth of the genetically engineered bacteria GS115-Col(I+III) induced for 24h, 48h, and 72h (all 5-fold dilutions));

[0033] Figure 4 This is a graph showing the concentration test results of collagen Col(I+III) expressed by genetically engineered bacteria GS115-Col(I+III) at different induction times;

[0034] Figure 5Figure 1 shows the electrophoresis results of protein purification by SP strong cationic chromatography of the supernatant of high-density fermentation induction of genetically engineered bacteria GS115-Col(I+III) (1: fermentation supernatant; 2: fermentation supernatant diluted 5 times (load sample); 3: flow-through after SP purification; 4: target protein eluted in elution buffer 1; 5: sample eluted in elution buffer 2);

[0035] Figure 6 This is a graph showing the results of the recombinant collagen Col(I+III) in this application promoting cell proliferation. DETAILED DESCRIPTION

[0036] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below with reference to the accompanying drawings and specific embodiments.

[0037] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all the same.

[0038] The following materials are involved in the following examples:

[0039] YPD liquid culture medium includes the following components: 10 g / L yeast powder, 20 g / L peptone, and 20 g / L glucose.

[0040] YPD solid culture medium includes the following components: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, and 2% agar powder.

[0041] BMGY liquid medium includes the following components: 10 g / L yeast powder, 20 g / L peptone, 100 mM potassium phosphate buffer (pH 6.0), 13.4 g / L YNB, (4 × 10 -5 )% biotin and 10g / L glycerol.

[0042] BMMY liquid medium includes the following components: 10 g / L yeast powder, 20 g / L peptone, 100 mM potassium phosphate buffer (pH 6.0), 13.4 g / L YNB, (4 × 10 -5 )% biotin and 1% methanol.

[0043] BSM basal salt medium includes the following components: 26.7 ml / L of 85% phosphoric acid, 0.93 g / L of calcium sulfate, 18.2 g / L of potassium sulfate, 14.9 g / L of magnesium sulfate heptahydrate, 4.13 g / L of potassium hydroxide, 40 ml / L of glycerol and 4.35 ml / L of PTM1 trace elements.

[0044] PMT1 medium includes the following components: 6 g / L copper sulfate pentahydrate, 0.08 g / L sodium iodide, 3 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride hexahydrate, 20 g / L zinc chloride, 65 g / L ferrous sulfate, and 0.2 g / L biotin.

[0045] Prestained protein markers were purchased from Thermo Fisher Scientific, and the host strain Pichia pastoris GS115 was purchased from Beyotime Biotechnology Co., Ltd.

[0046] SP Bestarose FF strong cationic chromatography medium was purchased from Boglon Biotechnology Co., Ltd.

[0047] Example 1. Construction of recombinant collagen expression vector pPICZaA-Col(I+III)

[0048] The advantage of using Pichia pastoris to express collagen is that the protein can be secreted extracellularly, reducing cellular stress and lowering production costs. However, a serious problem during the fermentation process is protein degradation. This is because Pichia pastoris itself secretes proteases during fermentation that may degrade human-like collagen.

[0049] To reduce protein degradation and increase protein yield, this application used computer intelligent computing tools to assist in analyzing the stability of specific cell surface receptor binding domains and peptides in these regions on type I and type III collagen. This application avoided common protease cleavage sequences (such as Kex2) and obtained the following specific peptides on human type I and type III collagen (the recombinant collagen of this application). The sequence of the basic structural unit of the recombinant collagen is as follows:

[0050] (1) The amino acid sequence of recombinant collagen Col(I+III) is shown in SEQ ID NO: 1:

[0051] GPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGPQGIAGQRGVVGLPGQRGERGFPGLPGLKGENGLPGENGAPGPMGPRGAPGERGRPGAPGPCCGG (SEQ ID NO: 1).

[0052] (2) The nucleotide sequence of recombinant collagen Col(I+III) is shown in SEQ ID NO: 2:

[0053]

[0054] (3) The nucleotide sequence of recombinant collagen Col(I+III) was optimized based on the codon preference of Pichia pastoris, and the DNA sequence of recombinant collagen Col(I+III) was synthesized by whole gene. The synthesized DNA sequence was cloned into the pPICZaA plasmid, and the pPICZaA-Col(I+III) expression vector was successfully constructed.

[0055] The nucleotide sequence of the DNA sequence encoding the recombinant collagen Col (I+III) after nucleotide sequence optimization is shown in SEQ ID NO: 3:

[0056]

[0057] Example 2: Screening of high-copy strains of recombinant collagen Col(I+III)

[0058] 1. Preparation of Pichia pastoris GS115 competent cells: For detailed preparation procedures, see the (Pichia pastoris expression manual).

[0059] 2. Extraction and linearization of the expression vector pPICZaA-Col(I+III) plasmid: For details on the extraction process, see the third edition of Molecular Cloning Protocols; written by J. Shambrook, translated by Huang Peitang.

[0060] 3. Use the expression vector pPICZaA-Col(I+III) to transform Pichia pastoris GS115 competent cells using an electroporator. For details on the electroporator transformation process, see the (Pichia pastoris Expression Operation Manual).

[0061] 4. Screening for high-copy strains: Linearize pPICZaA-Col(I+III) using the restriction endonuclease SacI. A clear electrophoretic band at the theoretical molecular weight of 4500 bp should be observed. Transform the plasmid into the competent Pichia pastoris host strain GS115 using an electroporator. Plate the electroporated bacterial solution onto YPD solid medium plates and incubate at 30°C for 2–5 days until a single colony (positive transformant) appears. A single colony was selected and inoculated into a YPD (containing 100 μg / ml bleomycin) liquid culture medium at 30°C and 220 rpm, and then transferred to a BMGY (containing 100 μg / ml bleomycin) liquid culture medium. After culturing at 30°C and 220 rpm for 24 hours, the supernatant was removed and collected by centrifugation at 5000 rpm for 10 minutes. The BMGY liquid culture medium was finally replaced with BMMY (no antibiotics added) liquid culture medium, and the inducer 1% (V / V) methanol was added every 24 hours at 30°C and 220 rpm for induction. The fermentation supernatant was taken every day for three consecutive days for SDS-PAGE gel electrophoresis (5% stacking gel, 12% separating gel). A clear electrophoretic band at the theoretical molecular weight of 35 kDa indicated that the protein had been expressed. The strain with the highest protein expression was selected and named GS115-Col(I+III).

[0062] The results are as follows Figures 1-2 As shown, the electrophoresis diagram of nucleic acid extracted from the non-linearized and SacI-linearized pPICZaA-Col(I+III) plasmids is shown in Figure 1 As shown; the induced expression of single clones selected after electroporation of the non-transfected Pichia pastoris host strain GS115 and the pPICZaA-Col (I+III) plasmid in GS115 competent cells is shown as follows: Figure 2 shown.

[0063] Example 3: High-density fermentation of recombinant collagen Col(I+III)

[0064] The genetically engineered bacteria GS115-Col (I+III) screened in Example 4 was inoculated into 50 mL of YPD (containing 100 μg / ml bleomycin) liquid medium and cultured at 30°C, 220 rpm for 24 h, then transferred to 300 mL of BMGY (containing 100 μg / ml bleomycin) liquid medium and cultured at 30°C, 220 rpm until the OD 600 10 to 15 is used as fermentation seed liquid.

[0065] Before inoculation, adjust the pH of the BSM culture medium in the 10L fermenter to 5.0 with ammonia water, and then add 17.4ml of PTM1 trace element solution. Inoculate the fermentation seed liquid into the fermenter at a 10% inoculation rate, adjust the ventilation volume to 4L / min, the stirring speed to 500rpm, and calibrate the dissolved oxygen value at this time to 100%; after culturing for 20 hours, the carbon source in the culture medium is consumed, the DO value increases, and 50% glycerol (containing PTM1 12ml / L) is started to be added. Control the dissolved oxygen to maintain DO at 20-30% until the bacterial OD 600 At 200-250°C, stop adding glycerol and adjust the pH to 6.0. Start induction with methanol (containing 12 ml / L of PTM1). Initially, control the dissolved oxygen to maintain the DO above 70%. After 4 hours, gradually increase the methanol flow rate to control the dissolved oxygen to maintain the DO between 20-30%. The maximum methanol residual should not exceed 10 g / L. Every 24 hours after induction, collect the fermentation supernatant for SDS-PAGE gel electrophoresis (5% stacking gel, 12% separating gel) and perform protein concentration detection (BCA assay kit).

[0066] The electrophoresis results of the genetically engineered bacteria GS115-Col(I+III) after high-density fermentation induction expression are shown in the figure below. Figure 3 As shown in the figure, the concentration test results of collagen Col(I+III) expressed by genetically engineered bacteria GS115-Col(I+III) at different induction times are as follows Figure 4 As shown, the total protein expression concentrations corresponding to 24h, 48h, and 72h of induction were 0.47mg / ml, 1.2mg / ml, and 2mg / ml, respectively.

[0067] Example 4: Purification of recombinant collagen Col(I+III)

[0068] The fermentation supernatant obtained by high-density fermentation of the genetically engineered bacteria GS115-Col(I+III) in Example 3 was purified by SP strong cationic chromatography.

[0069] Take 200ml of the supernatant and adjust the pH to 4.0 with phosphoric acid, then dilute 5 times with pure water to 2000ml, with a conductivity of 9.2mS / cm; centrifuge at 9000rpm for 20min, filter through a 0.45um membrane, and load onto a SP strong cation chromatography column with a bed volume of 100ml at a flow rate of 5ml / min; wash back to the baseline with equilibration buffer (20mM phosphate buffer, pH4.0) at a flow rate of 8ml / min; then elute with elution buffer 1 (20mM phosphate buffer, pH7.0, containing 300mM NaCl) and elution buffer 2 (20mM phosphate buffer, pH7.0, containing 1000mM NaCl) at a flow rate of 8ml / min. The purified sample was subjected to SDS-PAGE gel electrophoresis (5% stacking gel, 12% separation gel) to obtain the recombinant collagen Col (I+III) of the present application.

[0070] The supernatant of the genetically engineered bacteria GS115-Col (I+III) in Example 3 was induced by high-density fermentation and purified by SP strong cationic chromatography. The results of protein electrophoresis were as follows: Figure 5 shown.

[0071] Example 5: Recombinant collagen Col(I+III) promotes cell proliferation

[0072] The experiment of promoting cell proliferation includes the following steps:

[0073] (1) BALB / c 3T3 cells were seeded in 96-well cell culture plates containing DMEM medium (1×10 5 ~5×10 5 ), cultured in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0074] (2) Change to serum-free DMEM medium and continue culturing for 12 h.

[0075] (3) Add the sample to be tested and continue culturing for 24 to 48 hours.

[0076] (4) Add 10 μl of CCK-8 reagent to each well and incubate in a cell culture incubator at 37°C and 5% CO2 for 2 h before removing the cells.

[0077] (5) Use a microplate reader to read the absorbance values ​​of the 96-well plate at 450 nm and 630 nm. Take 630 nm as the reference wavelength and measure the absorbance at 450 nm. Record the measurement results and then use the formula: cell proliferation rate = [(sample OD - blank control OD) / (blank control OD - culture medium OD)] × 100%.

[0078] like Figure 6As shown, the experimental results show that: at a concentration of 80 μg / mL, the recombinant collagen Col (I+III) can promote the maximum proliferation rate of fibroblasts to 41.3%.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application 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 application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

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

1.

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

3.

3. An expression vector, characterized in that The expression vector comprises a nucleic acid molecule encoding the recombinant collagen as claimed in claim 2.

4. The expression vector according to claim 3, wherein The expression vector is pPICZaA plasmid.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the expression vector according to claim 3 or 4.

6. The genetically engineered bacterium according to claim 5, wherein The genetically engineered bacteria is Pichia pastoris.

7. The method for expressing recombinant collagen in Pichia pastoris according to claim 1, wherein The following steps are involved: S1. Linearizing the nucleic acid molecule encoding the recombinant collagen protein according to claim 1 and the Pichia pastoris expression vector pPICZaA using restriction endonucleases, transforming the linearized fragments into competent Pichia pastoris host bacteria GS115, and culturing them. Single colonies are selected for cultivation, and the fermentation supernatant is then subjected to agarose gel electrophoresis to select strains expressing the recombinant collagen protein. S2. Inoculate the strain obtained in step S1 into a liquid culture medium to obtain a fermentation seed solution; The fermentation seed liquid was inoculated into the fermentation tank for cultivation, glycerol was added, and the dissolved oxygen was controlled to maintain DO at 20-30% until the bacterial OD 600 At 200-250°C, stop adding glycerol; S3. After stopping the flow addition of glycerol, start the flow addition of methanol for induction, control the dissolved oxygen to maintain the DO at 20-30%, obtain the fermentation broth, and then perform agarose gel electrophoresis for separation and purification.

8. The method for expressing recombinant collagen in Pichia pastoris according to claim 7, wherein: In step S1, if agarose gel electrophoresis shows a clear electrophoretic band at the 35 kDa position, it means that the recombinant collagen protein according to claim 1 has been expressed.

9. The method for expressing recombinant collagen in Pichia pastoris according to claim 7, wherein: The selection of single bacterial colonies for cultivation comprises the following steps: Select a single colony and inoculate it into YPD liquid medium and culture it at 30℃ and 220rpm. Then transfer it to BMGY liquid medium and culture it at 30℃ and 220rpm for 24h. Then centrifuge it at 5000 rpm for 10 minutes, pour out the supernatant to collect the bacteria, and finally replace the BMGY liquid medium with BMMY liquid medium. Then add inducer and induce it at 30℃ and 220rpm, and take the fermentation supernatant.

10. The method for expressing recombinant collagen in Pichia pastoris according to claim 7, wherein: In step S3, the methanol induction time is 24 to 72 hours.

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