Recombinant collagen and expression method thereof in pichia pastoris

Abstract

The invention relates to the technical field of biology, and particularly discloses recombinant collagen and an expression method thereof in pichia pastoris. The amino acid sequence of the basic structural unit of the recombinant collagen is shown as SEQ ID NO: 1, and the nucleotide sequence of the basic structural unit is shown as SEQ ID NO: 2. The recombinant collagen Col (I + III) contains a specific basic structure unit, so that the expression quantity of the collagen in host cells can be increased, the stability and activity of the collagen can be improved, and the proliferation of fibroblasts can be remarkably promoted; moreover, by adopting the genetic engineering mode of the collagen, the expression quantity of the recombinant collagen in host cells can be better improved, and the proliferation of fibroblasts is promoted.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant collagen and an expression method thereof 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 mainly comes from two sources: collagen extracted from animal tissues and recombinant collagen prepared by recombinant DNA technology. Collagen extracted from animal tissues can have a complete collagen molecular structure, but there is also a risk of introducing viruses, and there is also a certain immunogenicity. Collagen prepared by recombinant DNA technology has better biocompatibility, water solubility, and stability, and there is no risk of introducing viruses.

[0004] The U.S. Food and Drug Administration (FDA) has recognized Pichia pastoris as a generally regarded as safe (GRAS) microorganism, which is widely used in the fields of medicine and food. The Pichia pastoris expression system has the advantages of less secretion of impurities, low culture cost, and mature fermentation technology. A variety of exogenous proteins have been successfully expressed in this system, including epidermal augmentum factor (EGF), human serum albumin (HSA), hepatitis B surface antigen (HBsAg), vaccines, insulin products, enzyme preparations, etc. In addition, Pichia pastoris is also widely used as a host cell for the food industry and biomaterial expression. For example, cellulase, β-mannanase, hemicellulase, etc. 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 a correct folding structure during the preparation of 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 it in Pichia pastoris.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

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

[0009] The inventors of the present application have found through extensive research and experiments 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 highest proliferation rate of fibroblasts promoted by recombinant collagen Col (I+III) is 41.3%.

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

[0012] The present application provides an expression vector, which comprises 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 pPICZaA plasmid.

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

[0015] The present application provides a genetically engineered bacterium, wherein the genetically engineered bacterium comprises 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. The 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, and the linearized fragments are transformed into the competent medium of the Pichia pastoris host bacteria GS115 for cultivation, and a single colony is selected for cultivation, and the fermentation supernatant is taken 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 culture, 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 induction was started, and the dissolved oxygen was controlled to maintain DO at 20-30%, and the fermentation liquid was obtained, and then agarose gel electrophoresis was performed for separation and purification.

[0021] Collagen is prepared through genetic engineering, which requires the selection of appropriate collagen domains and the rational optimization of the amino acid sequence of the selected collagen domains in order to increase the expression of collagen in host cells, as well as 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, agarose gel electrophoresis shows an obvious electrophoretic band at the 35 kDa position, indicating 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 of the present application, the step of selecting 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℃, 220rpm. Then transfer it to BMGY liquid medium and culture it at 30℃, 220rpm for 24h. Then centrifuge it at 5000rpm for 10 minutes, pour out the supernatant and collect the bacteria. Finally, replace the BMGY liquid medium with BMMY liquid medium, add the inducer and induce it at 30℃, 220rpm, and take the fermentation supernatant.

[0025] As a preferred embodiment of the method for expressing recombinant collagen in Pichia pastoris of 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, indicating 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 and a method for expressing it in Pichia pastoris. The recombinant collagen Col (I+III) of the present application contains specific basic structural units, which can increase the expression level of collagen in host cells, as well as the stability and activity of collagen, thereby significantly promoting the proliferation of fibroblasts; and, the preparation of the collagen of the present application by genetic engineering can better increase the expression level of recombinant collagen 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 or induced expression of single clones of Pichia pastoris GS115 (1: Pichia pastoris GS115 without plasmid transfer, 2-9: induced expression of different single clones selected after electroporation of pPICZaA-Col(I+III) plasmid into competent cells of Pichia pastoris GS115);

[0032] Figure 3 The electrophoresis results of high-density fermentation induced expression of genetically engineered bacteria GS115-Col (I+III) (1: 5-fold dilution of the fermentation supernatant of genetically engineered bacteria GS115-Col (I+III) without induction; 2-4: protein electrophoresis results of the fermentation supernatant of genetically engineered bacteria GS115-Col (I+III) induced for 24h, 48h, and 72h (all 5-fold dilution));

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

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

[0036] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with 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 of the same type.

[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 host bacteria Pichia pastoris GS115 were purchased from Bio-Technology 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 outside the cell, reducing cell stress and lowering production costs. However, a serious problem faced during the fermentation process is protein degradation, because Pichia pastoris itself secretes proteases during the fermentation process, which may degrade human-like collagen.

[0049] In order to reduce protein degradation and increase protein yield, the present application uses computer intelligent calculation tools to assist in analyzing the stability of specific cell surface receptor binding domains and regional peptides on type I and type III collagen. The present application avoids the common protease (Kex2, etc.) cleavage sequence and obtains the following specific peptides on human type I and type III collagen (recombinant collagen of the present 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 the recombinant collagen Col(I+III) was optimized based on the codon preference of Pichia pastoris, and the DNA sequence of the 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 details of the preparation process, see (Pichia pastoris expression operation manual).

[0059] 2. Extraction of expression vector pPICZaA-Col(I+III) plasmid and linearization of plasmid: For details of the extraction process, please refer to the third edition of Molecular Cloning Experiment Guide; written by J. Shambrook [USA] and 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 (Pichia pastoris Expression Operation Manual).

[0061] 4. High copy strain screening: pPICZaA-Col (I+III) was linearized by restriction endonuclease SacI, and an obvious electrophoresis band was observed at the theoretical molecular weight of 4500bp. Then, the strain was transformed into the competent state of Pichia pastoris host bacteria GS115 by electroporation instrument, and the electroporated bacterial solution was spread on YPD solid culture medium plate, and inverted cultured at 30℃ for 2-5 days until a single colony (positive transformant) appeared. 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 for 24 hours at 30°C and 220 rpm, the supernatant was removed and the bacteria were collected by centrifugation at 5000 rpm for 10 minutes. Finally, the BMGY liquid culture medium was replaced with a 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% concentrated gel, 12% separation gel). The presence of an obvious 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 of nucleic acid extracted from the unlinearized and SacI-linearized pPICZaA-Col(I+III) plasmids is shown in Figure 1 As shown; the induced expression of single clones selected after the untransfected Pichia yeast host strain GS115 and GS115 competent cells were electroporated with the pPICZaA-Col(I+III) plasmid as shown Figure 2 shown.

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

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

[0065] Before inoculation, adjust the pH of the BSM 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 20h of cultivation, the carbon source in the culture medium is consumed, the DO value increases, and 50% glycerol (containing PTM1 12ml / L) is started. Control the dissolved oxygen to maintain DO at 20-30% until the bacterial OD 600 At 200-250, stop adding glycerol and adjust pH to 6.0. Start adding methanol for induction (containing PTM1 12ml / L). At the beginning of methanol induction, control the dissolved oxygen to maintain DO above 70%. After 4 hours, gradually increase the methanol flow rate to control the dissolved oxygen to maintain DO at 20-30%. The maximum methanol residue should not exceed 10g / L. Every 24 hours after induction, take the fermentation supernatant for SDS-PAGE gel electrophoresis (5% concentrated gel, 12% separation gel) and protein concentration detection (BCA assay kit).

[0066] The electrophoresis results of the genetically engineered bacteria GS115-Col(I+III) after high-density fermentation and 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 cation chromatography.

[0069] Take 200ml of supernatant and adjust the pH to 4.0 with phosphoric acid, then dilute 5 times with pure water to 2000ml, the conductivity is 9.2mS / cm; 9000rpm, 20min centrifugation 0.45um membrane filtration, load to the SP strong cation chromatography column with a column bed volume of 100ml, the flow rate is 5ml / min; balance buffer (20mM phosphate buffer, pH4.0) wash back to the baseline, the flow rate is 8ml / min; then elute through elution buffer1 (20mM phosphate buffer, pH7.0, containing 300mM NaCl) and elution buffer2 (20mM phosphate buffer, pH7.0, containing 1000mM NaCl), the flow rate is 8ml / min. The purified sample was identified by SDS-PAGE gel electrophoresis (5% concentrated 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 cation chromatography. The results of protein electrophoresis are 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 of the 96-well plate at 450 nm and 630 nm. Take 630 nm as the reference wavelength, 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 solution of the present application rather than 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, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A recombinant collagen, characterized in that: The amino acid sequence of the basic structural unit of the recombinant collagen is shown in SEQ ID NO: 1, and the nucleotide sequence of the basic structural unit is shown in SEQ ID NO:

2.

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

3.

3. An expression vector, characterized in that: The expression vector comprises a DNA sequence encoding the recombinant collagen as claimed in claim 1.

4. The expression vector according to claim 3, characterized in that The expression vector includes pPICZaA plasmid.

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

6. The genetically engineered bacterium according to claim 5, characterized in that The genetically engineered bacteria include Pichia pastoris.

7. The method for expressing recombinant collagen in Pichia pastoris according to claim 1, characterized in that: The following steps are involved: S1. The DNA sequence encoding the recombinant collagen protein as claimed in claim 1 and the Pichia pastoris expression vector pPICZaA are linearized by restriction endonuclease digestion, and the linearized fragments are transformed into the competent state of the Pichia pastoris host bacteria GS115 for cultivation, and a single colony is selected for cultivation, and the fermentation supernatant is taken and then subjected to agarose gel electrophoresis to select the strain expressing the recombinant collagen protein; S2, inoculating the strain obtained in step S1 into a liquid culture medium for cultivation to obtain a fermentation seed liquid; 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, stop adding glycerol; S3. After stopping the flow addition of glycerol, methanol induction was started, and the dissolved oxygen was controlled to maintain DO at 20-30%, and the fermentation liquid was obtained, and then agarose gel electrophoresis was performed for separation and purification.

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

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

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

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