Construction method of collagen expression system with adjustable hydroxylation degree

Abstract

The invention relates to a construction method of a collagen expression system with adjustable hydroxylation degree, which comprises the following steps: optimizing a humanized I-type collagen sequence according to pichia pastoris codon characteristics, synthesizing a recombinant I-type humanized collagen DNA sequence, carrying out double enzyme digestion on the recombinant I-type humanized collagen DNA sequence and plasmid 1, and connecting to obtain a recombinant collagen expression vector; optimizing according to the codon characteristics of pichia pastoris to obtain a proline hydroxylase DNA sequence, carrying out double enzyme digestion on the proline hydroxylase DNA sequence and a recombinant plasmid 2, and connecting to obtain a proline hydroxylase free expression vector; a recombinant collagen expression vector is linearized and then electrically transformed into pichia pastoris, a proline hydroxylase free expression vector is electrically transformed into the pichia pastoris, and the collagen expression system with the adjustable hydroxylation degree is obtained. The collagen with different hydroxylation degrees can be obtained through the constructed expression system, the method is suitable for various application scenes, the production efficiency is improved, the production cost is saved, and the method has important practical significance and wide market prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for constructing a collagen expression system with adjustable hydroxylation degree. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of the total protein. It is closely related to the formation, maturation, transmission of intercellular information, joint lubrication, wound healing, calcification, blood coagulation and aging of tissues. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics and food industries. At present, the main methods for producing collagen are traditional extraction, chemical synthesis and modern biotechnology production. The collagen required for research is generally extracted from animal skin and Achilles tendon and other tissues, and most of them are easy to handle bovine tendon and rat tail tendon as raw materials. The extracted animal collagen has potential viral risks and is easy to induce immune response. There are also problems such as difficult extraction process, long cycle, high cost and poor water solubility. The use of genetic engineering technology to express recombinant collagen has the advantages of low cost and high efficiency. Compared with animal collagen extracted by traditional extraction methods, recombinant human collagen has the advantages of high yield, batch stability, strong hydrophilicity, good biocompatibility and no immune virus risk, and is an ideal biomedical material. Hydroxyproline is a non-natural amino acid unique to collagen. Its expression plays an important role in the formation of the helical domain of collagen and the stability of its structure. It is one of the main differences between recombinant collagen and natural collagen.

[0003] The yeast expression system combines the advantages of prokaryotic expression, such as short cycle, low cost, and easy operation, and is easy to scale up for industrial level culture; at the same time, it has the ability of eukaryotic cells to perform spatial folding and glycosylation modification of proteins, which greatly improves the solubility of proteins. The exogenous gene expression system using Pichia pastoris as a host has developed rapidly in recent years and is considered an important and widely accepted tool for producing recombinant proteins in molecular biology. Pichia pastoris heterologous expression is mainly based on integrated expression, and the degree of hydroxylation of the expressed collagen is single, which is difficult to meet the application of multiple scenarios.

[0004] Therefore, it is of great value and significance to provide a method for constructing a collagen expression system with adjustable degree of hydroxylation to meet the application needs of various scenarios. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing a collagen expression system with controllable hydroxylation degree.

[0006] To solve the above problems, the technical solution adopted by the present invention is: The present invention provides a method for constructing a collagen expression system with a controllable hydroxylation degree, comprising the following steps: S1: Optimize the humanized type I collagen nucleic acid sequence according to the codon characteristics of Pichia pastoris, synthesize the recombinant type I humanized collagen DNA sequence, and then double-digest and connect it with plasmid 1 to obtain the recombinant collagen expression vector; S2: Optimizing the proline hydroxylase DNA sequence according to the codon characteristics of Pichia pastoris; double-digesting and connecting the recombinant plasmid 2 and the proline hydroxylase DNA sequence to obtain a proline hydroxylase free expression vector; S3: After linearization, the recombinant collagen expression vector is electro-transformed into Pichia pastoris; the proline hydroxylase free expression vector is electro-transformed into the Pichia pastoris containing the recombinant collagen expression vector DNA sequence to obtain a collagen expression system with a controllable degree of hydroxylation.

[0007] As a further embodiment of the present invention, the recombinant humanized type I collagen DNA sequence is shown as SEQ ID No.1.

[0008] As a further embodiment of the present invention, the proline hydroxylase DNA sequence is shown as SEQ ID No.2.

[0009] As a further embodiment of the present invention, the plasmid 1 is plasmid pPIC9K; the Pichia pastoris is Pichia pastoris GS115.

[0010] As a further embodiment of the present invention, the recombinant plasmid 2 is obtained by linking three DNA fragments having DNA sequences of SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 respectively.

[0011] As a further embodiment of the present invention, the DNA fragment with the sequence shown in SEQ ID No.3 is obtained by extension primer 1 and extension primer 2 using the DNA sequence of pPICZαA plasmid as a template; the sequence of extension primer 1 is shown in SEQ ID No.6; and the sequence of extension primer 2 is shown in SEQ ID No.7.

[0012] As a further embodiment of the present invention, the DNA fragment with the sequence shown in SEQ ID No. 4 is synthesized according to the GenBank: M11199.1 sequence in NCBI.

[0013] As a further embodiment of the present invention, the DNA fragment with the sequence shown in SEQ ID No.5 is obtained by extension primer 3 and extension primer 4 using the DNA sequence of pet30a vector as a template; the sequence of extension primer 3 is shown in SEQ ID No.8; and the sequence of extension primer 4 is shown in SEQ ID No.9.

[0014] As a further embodiment of the present invention, the restriction endonucleases used in the double enzyme digestion process are EcoR I and Not I; the restriction endonuclease used in the linearization process of S3 is Sac I.

[0015] As a further embodiment of the present invention, the hydroxylation degree is regulated by changing the dosage of the inducer IPTG.

[0016] The beneficial effects of adopting the above technical solution are: 1. Based on the existing Pichia pastoris expression system, the present invention provides a new method for constructing an expression vector, constructs a plasmid that can be freely expressed in Pichia pastoris, loads the proline hydroxylase expression gene, and realizes the controllable expression of proline hydroxylase.

[0017] 2. The expression system obtained by the construction method provided by the present invention can regulate the expression amount of the gene by controlling the fermentation conditions of the recombinant Pichia pastoris, thereby obtaining collagen with different degrees of hydroxylation.

[0018] 3. The expression system obtained by the construction method provided by the present invention can controllably express collagen with different degrees of hydroxylation, thereby meeting the use of various application scenarios, improving collagen production efficiency, and saving production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of the structure of the expression vector pPIC9K-COL1A2 of Example 1 of the present invention; Figure 2 is a schematic diagram of the structure of the expression vector pPICZα A-pars1-t7-lacI in Example 1 of the present invention; Figure 3 is a schematic diagram of the structure of the expression vector pPICZα A-pars1-t7-lacI-P4H in Example 1 of the present invention; Figure 4 It is the electrophoresis diagram of the fermentation samples obtained in Examples 2-5 of the present invention; wherein, lane 1 is the fermentation sample of Example 2, lane 2 is the fermentation sample of Example 3, lane 3 is the fermentation sample of Example 4, and lane 4 is the fermentation sample of Example 5. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0021] Example 1 S1. Construction of recombinant humanized collagen expression vector pPIC9K-COL1A2: (1) The desired amino acid sequence was obtained from the triple helix region of the nucleic acid sequence of humanized type I collagen in GenBank: KU177988.1 in NCBI, and after optimization based on the codon characteristics of the Pichia pastoris expression system, it was synthesized by Nanjing GenScript Biotechnology Co., Ltd. to obtain the recombinant humanized type I collagen DNA sequence shown in SEQ ID No. 1; (2) Add 10 μL of recombinant humanized collagen type I DNA sequence or 10 μL of pPIC9K to 4 μL 10Xfastdigest buffer, 4 μL EcoR I, 4 μL Not I and 18 μL sterile water, and react at 37°C for 10 min to obtain DNA fragments digested with EcoR I and Not I, respectively; add 1 μL of double-digested plasmid pPIC9K and 4 μL of digested recombinant humanized collagen type I DNA sequence to 2 μL T4 DNA ligase, and react at 22°C for 60 min to obtain the recombinant humanized collagen expression vector pPIC9K-COL1A2; S2. Construction of proline hydroxylase free expression vector pPICZαA-pars1-t7-lacI-P4H: (1) Based on the amino acid sequence of YP_010777505.1 in NCBI, the proline hydroxylase DNA sequence shown in SEQ ID No. 2 was obtained after optimizing the codon characteristics of the Pichia pastoris expression system; (2) Add 2 μL of pPICZαA, 2 μL of extension primer 1 and 2 μL of extension primer 2 to 25 μL of PrimeSTAR MaxVer.2 Premix (2X) and 19 μL of sterile water, place in a PCR instrument, set the reaction conditions, the reaction conditions are shown in Table 1 below, and obtain a DNA sequence as shown in SEQ ID No. 3, which retains the original replicon and bleomycin resistance expression part; Table 1 PCR reaction conditions for the DNA sequence shown in SEQ ID No.3 (3) Synthesize the yeast replicon DNA sequence shown as SEQ ID No. 4 based on the GenBank: M11199.1 sequence in NCBI; (4) Add 2 μL of pet30a, 2 μL of extension primer 3 and 2 μL of extension primer 4 to 25 μL of PrimeSTAR MaxVer.2 Premix (2X) and 19 μL of sterile water, place in a PCR instrument, set the reaction conditions, the reaction conditions are shown in Table 2 below, and obtain the DNA sequence shown in SEQ ID No. 5, which retains the nucleic acid sequence of the T7 promoter, the multiple cloning site and the lactose operon repressor protein lacI; Table 2 PCR reaction conditions for the DNA sequence shown in SEQ ID No.5 (6) The three DNA fragments with sequences as shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 were seamlessly connected according to the instructions attached to the GeneArt™ Gibson Assembly® EX Cloning Kits reagent of Thermo Company, and then transformed into Escherichia coli for expression to obtain the recombinant plasmid pPICZαA-pars1-t7-lacI; (7) Add 10 μL of proline hydroxylase DNA sequence or 10 μL of recombinant plasmid pPICZαA-pars1-t7-lacI to 4 μL 10X fastdigest buffer, 4 μL EcoR I, 4 μL Not I and 18 μL sterile water, and react at 37°C for 10 min to obtain the proline hydroxylase DNA fragment and the recombinant plasmid pPICZαA-pars1-t7-lacI digested by EcoR I and Not I, respectively; add 4 μL of the proline hydroxylase DNA sequence and 2 μL of the recombinant plasmid pPICZαA-pars1-t7-lacI digested by EcoR I and Not I to 2 μL T4 DNA ligase to obtain the proline hydroxylase free expression vector pPICZαA-pars1-t7-lacI-P4H; S3. Construction of collagen-expressing recombinant Pichia pastoris GS115-COL1A2-P4H: Add 2 μL of recombinant humanized collagen expression vector pPIC9K-COL1A2 to 1 μL of restriction endonuclease Sal I, react at 37°C for 10 min to obtain the linearized DNA fragment of pPIC9K-COL1A2; add 5 μL of the linearized DNA fragment of pPIC9K-COL1A2 to 80 μL of Pichia pastoris GS115 competent cells, gently pipette to mix, transfer to a 2 mm electroporation cup, incubate on ice for 2 min, electroporate at 2.0 kV for 6 ms, and immediately add 900 μL YPD medium, incubate at 30℃ shaker for 3h, spread on G418-resistant YPD plate medium, and culture at 30℃ for 48h to obtain recombinant Pichia pastoris GS115-COL1A2 capable of expressing recombinant type I humanized collagen; add 3μL of proline hydroxylase free expression vector pPICZαA-pars1-t7-lacI-P4H to 80μL of recombinant Pichia pastoris GS115-COL1A2 competent cells, gently blow to mix, transfer to a 2mm electric shock cup, incubate on ice for 2min, electroporate at 2.0kV for 6ms, immediately add 900μL YPD medium, incubate at 30℃ shaker for 3h, spread on bleomycin-resistant YPD plate medium, and culture at 30℃ for 48h to obtain collagen-expressing recombinant Pichia pastoris GS115-COL1A2-P4H.

[0022] Example 2 S1, fermentation: add 2.5L fermentation medium into a 5L fermenter, sterilize and cool, inoculate recombinant Pichia at 10% inoculation, fermentation conditions: 30℃, pH=5.0, 20% dissolved oxygen fermentation, after the carbon source of the basal medium is consumed, feed glycerol medium and continue fermentation for 24h, stop feeding glycerol medium, add IPTG with a final concentration of 1.0mmol / L; after the dissolved oxygen rises to 60%, start feeding methanol induction medium and continue fermentation for 96h; Fermentation medium: MgSO4·7H2O 14.9 g / L, CaSO4 0.94 g / L, K2SO4 18.2 g / L, CaSO4 0.94 g / L, 85% concentrated H3PO4 26.7 mL / L, glycerol 40 g / L, PTM1 4.35 mL / L, KOH 4.13 g / L, sterilization at 121 °C for 20 min; PTM1: CuSO4·5H2O 6.00g / L, MnSO4·H2O 3.00g / L, ZnCl2 20.00g / L, NaI 0.088g / L, CoCl2 0.50g / L, Na2MoO4·2H2O 0.20g / L, H3BO4 0.02g / L, Biotin 0.20g / L, FeSO4·7H2O 65.00g / L, 5mL of concentrated sulfuric acid; Glycerol feed medium: 50% glycerol, 12 mL / L PTM1, sterilized at 121°C for 20 min for later use; Methanol-induced feed medium: Add PTM1 solution to 100% methanol to a final concentration of 12 mL / L; S2. Collagen purification: The fermentation broth was replaced with buffer 1 using an ultrafiltration membrane, and the obtained sample solution was loaded on Q-FF ion chromatography, equilibrated with buffer 1 for 2 times the column volume, and eluted with buffer 2, and samples were collected according to the peak position; Buffer 1: 20 mM Tris, pH = 8.0; Buffer 2: 20 mM Tris, 1 M NaCl, pH = 8.0; S3. Detection of collagen hydroproline content: Referring to the national standard GBT9695.23-2008 hydroxyproline detection method, the hydroxyproline content of the obtained collagen was detected, and the degree of proline hydroxylation was calculated to be 61%.

[0023] Example 3 S1, fermentation: add 2.5L fermentation medium into a 5L fermenter, sterilize and cool, inoculate recombinant Pichia at 10% inoculation, fermentation conditions: 30℃, pH=5.0, 20% dissolved oxygen fermentation, after the carbon source of the basal medium is consumed, feed glycerol medium and continue fermentation for 24h, stop feeding glycerol medium, add IPTG with a final concentration of 0.5mmol / L; after the dissolved oxygen rises to 60%, start feeding methanol induction medium and continue fermentation for 96h; Fermentation medium: MgSO4·7H2O 14.9 g / L, CaSO4 0.94 g / L, K2SO4 18.2 g / L, CaSO4 0.94 g / L, 85% concentrated H3PO4 26.7 mL / L, glycerol 40 g / L, PTM1 4.35 mL / L, KOH 4.13 g / L, sterilization at 121 °C for 20 min; PTM1: CuSO4·5H2O 6.00g / L, MnSO4·H2O 3.00g / L, ZnCl2 20.00g / L, NaI 0.088g / L, CoCl2 0.50g / L, Na2MoO4·2H2O 0.20g / L, H3BO4 0.02g / L, Biotin 0.20g / L, FeSO4·7H2O 65.00g / L, 5mL of concentrated sulfuric acid; Glycerol feed medium: 50% glycerol, 12 mL / L PTM1, sterilized at 121°C for 20 min for later use; Methanol-induced feed medium: Add PTM1 solution to 100% methanol to a final concentration of 12 mL / L; S2. Collagen purification: The fermentation broth was replaced with buffer 1 using an ultrafiltration membrane, and the obtained sample solution was loaded on Q-FF ion chromatography, equilibrated with buffer 1 for 2 times the column volume, and eluted with buffer 2, and samples were collected according to the peak position; Buffer 1: 20 mM Tris, pH = 8.0; Buffer 2: 20 mM Tris, 1 M NaCl, pH = 8.0; S3. Detection of collagen hydroproline content: Referring to the national standard GBT9695.23-2008 hydroxyproline detection method, the hydroxyproline content of the obtained collagen was detected, and the degree of proline hydroxylation was calculated to be 54%.

[0024] Example 4 S1, fermentation: add 2.5L fermentation medium into a 5L fermenter, sterilize and cool, inoculate recombinant Pichia at 10% inoculation, fermentation conditions: 30℃, pH=5.0, 20% dissolved oxygen fermentation, after the carbon source of the basal medium is consumed, feed glycerol medium and continue fermentation for 24h, stop feeding glycerol medium, add IPTG with a final concentration of 0.2mmol / L; after the dissolved oxygen rises to 60%, start feeding methanol induction medium and continue fermentation for 96h; Fermentation medium: MgSO4·7H2O 14.9 g / L, CaSO4 0.94 g / L, K2SO4 18.2 g / L, CaSO4 0.94 g / L, 85% concentrated H3PO4 26.7 mL / L, glycerol 40 g / L, PTM1 4.35 mL / L, KOH 4.13 g / L, sterilization at 121 °C for 20 min; PTM1: CuSO4·5H2O 6.00g / L, MnSO4·H2O 3.00g / L, ZnCl2 20.00g / L, NaI 0.088g / L, CoCl2 0.50g / L, Na2MoO4·2H2O 0.20g / L, H3BO4 0.02g / L, Biotin 0.20g / L, FeSO4·7H2O 65.00g / L, 5mL of concentrated sulfuric acid; Glycerol feed medium: 50% glycerol, 12 mL / L PTM1, sterilized at 121°C for 20 min for later use; Methanol-induced feed medium: Add PTM1 solution to 100% methanol to a final concentration of 12 mL / L; S2. Collagen purification: The fermentation broth was replaced with buffer 1 using an ultrafiltration membrane, and the obtained sample solution was loaded on Q-FF ion chromatography, equilibrated with buffer 1 for 2 times the column volume, and eluted with buffer 2, and samples were collected according to the peak position; Buffer 1: 20 mM Tris, pH = 8.0; Buffer 2: 20 mM Tris, 1 M NaCl, pH = 8.0; S3. Detection of collagen hydroproline content: Referring to the national standard GBT9695.23-2008 hydroxyproline detection method, the hydroxyproline content of the obtained collagen was detected, and the degree of proline hydroxylation was calculated to be 22%.

[0025] Example 5 S1, fermentation: add 2.5L fermentation medium into a 5L fermenter, sterilize and cool, inoculate recombinant Pichia at 10% inoculation, fermentation conditions: 30℃, pH=5.0, 20% dissolved oxygen fermentation, after the carbon source of the basal medium is consumed, feed glycerol medium and continue fermentation for 24h, stop feeding glycerol medium, do not add IPTG; after the dissolved oxygen rises to 60%, start feeding methanol induction medium and continue fermentation for 96h; Fermentation medium: MgSO4·7H2O 14.9 g / L, CaSO4 0.94 g / L, K2SO4 18.2 g / L, CaSO4 0.94 g / L, 85% concentrated H3PO4 26.7 mL / L, glycerol 40 g / L, PTM1 4.35 mL / L, KOH 4.13 g / L, sterilization at 121 °C for 20 min; PTM1: CuSO4·5H2O 6.00g / L, MnSO4·H2O 3.00g / L, ZnCl2 20.00g / L, NaI 0.088g / L, CoCl2 0.50g / L, Na2MoO4·2H2O 0.20g / L, H3BO4 0.02g / L, Biotin 0.20g / L, FeSO4·7H2O 65.00g / L, 5mL of concentrated sulfuric acid; Glycerol feed medium: 50% glycerol, 12 mL / L PTM1, sterilized at 121°C for 20 min for later use; Methanol-induced feed medium: Add PTM1 solution to 100% methanol to a final concentration of 12 mL / L; S2. Collagen purification: The fermentation broth was replaced with buffer 1 using an ultrafiltration membrane, and the obtained sample solution was loaded on Q-FF ion chromatography, equilibrated with buffer 1 for 2 times the column volume, and eluted with buffer 2, and samples were collected according to the peak position; Buffer 1: 20 mM Tris, pH = 8.0; Buffer 2: 20 mM Tris, 1 M NaCl, pH = 8.0; S3. Detection of collagen hydroproline content: refer to the national standard GBT9695.23-2008 hydroxyproline detection method, detect the hydroxyproline content of the obtained collagen, and calculate the degree of proline hydroxylation as 0%.

[0026] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a collagen expression system with adjustable hydroxylation degree, characterized in that: The following steps are involved: S1: Optimize the humanized type I collagen nucleic acid sequence according to the codon characteristics of Pichia pastoris, synthesize the recombinant type I humanized collagen DNA sequence, and then double-digest and connect it with plasmid 1 to obtain the recombinant collagen expression vector; S2: Optimizing the proline hydroxylase DNA sequence according to the codon characteristics of Pichia pastoris; double-digesting and connecting the recombinant plasmid 2 and the proline hydroxylase DNA sequence to obtain a proline hydroxylase free expression vector; S3: After linearization, the recombinant collagen expression vector is electro-transformed into Pichia pastoris; the proline hydroxylase free expression vector is electro-transformed into the Pichia pastoris containing the recombinant collagen expression vector DNA sequence to obtain a collagen expression system with a controllable degree of hydroxylation.

2. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The recombinant humanized type I collagen DNA sequence is shown in SEQ ID No.

1.

3. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The proline hydroxylase DNA sequence is shown as SEQ ID No.

2.

4. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The plasmid 1 is plasmid pPIC9K; the Pichia pastoris is Pichia pastoris GS115.

5. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The recombinant plasmid 2 is obtained by linking three DNA fragments whose DNA sequences are SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 respectively.

6. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 5, characterized in that: The DNA fragment with the sequence shown in SEQ ID No.3 is obtained by using extension primer 1 and extension primer 2 with the DNA sequence of pPICZαA plasmid as a template; the sequence of extension primer 1 is shown in SEQ ID No.6; The sequence of the extension primer 2 is shown as SEQ ID No.

7.

7. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 5, characterized in that: The DNA fragment with the sequence shown in SEQ ID No. 4 was synthesized according to the GenBank: M11199.1 sequence in NCBI.

8. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 5, characterized in that: The DNA fragment with the sequence shown in SEQ ID No.5 is obtained by extension primer 3 and extension primer 4 using the DNA sequence of pet30a vector as a template; the sequence of extension primer 3 is shown in SEQ ID No.8; and the sequence of extension primer 4 is shown in SEQ ID No.

9.

9. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The restriction endonucleases used in the double enzyme digestion process are EcoR I and Not I; the restriction endonuclease used in the linearization process of S3 is Sac I.

10. The method for constructing a collagen expression system with adjustable hydroxylation degree according to claim 1, characterized in that: The degree of hydroxylation is regulated by changing the amount of inducer IPTG.

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