A prokaryotic expression system and its preparation method and application

Abstract

The present invention discloses a prokaryotic expression system and a preparation method and application thereof. The prokaryotic expression system comprises prokaryotic cells expressing prolyl hydroxylase functional subunits P4HB and P4HA1 and transfer RNAs of codons AUA, AGG, AGA, CUA, CCC, GGA and CGG. The present invention co-expresses prolyl hydroxylase functional subunits P4HB and P4HA1 in the prokaryotic expression system. P4HB and P4HA1 are directed to recombinant collagen and can perform hydroxylation modification on proline in collagen, that is, prokaryotic cells can be enabled to perform post-translational hydroxylation modification on proline on amino acid chains, and more accurate recombinant collagen can be expressed; 7 rare codons are co-expressed, so that prokaryotic cells can obtain the ability to translate the amino acids corresponding to these codons, thereby increasing the expression amount of the protein and having broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a prokaryotic expression system and a preparation method and application thereof. Background Art

[0002] The content of many proteins in organisms is very small. In order to obtain a large amount of target protein, people use genetic engineering technology to introduce the nucleic acid sequence encoding the protein into a host cell to express it in large quantities. This method of inserting cloned genes into a suitable vector and then introducing them into host cells for expressing a large amount of protein is generally called protein expression technology. Protein expression technology has applications in protein purification, localization, protein domain analysis, and in vitro functional analysis of proteins.

[0003] According to the host cell, protein expression systems can be divided into two categories: prokaryotic expression systems and eukaryotic expression systems. Prokaryotic expression systems include Escherichia coli expression systems, Bacillus expression systems, Streptomyces expression systems, etc. Eukaryotic expression systems include yeast expression systems, insect expression systems, mammalian expression systems, and plant cell expression systems. Compared with other expression systems, the E. coli expression system has the advantages of clear genetic background, high expression level of target gene, short culture cycle, strong anti-pollution ability, easier growth and control, relatively simple experimental technology, low cost, a variety of E. coli strains and matching plasmids with various characteristics to choose from. For example, CN112029788A discloses a method for expressing and purifying a polypeptide using a prokaryotic expression system, wherein the method comprises: adding an enterokinase cleavage site to the C-terminus of a polypeptide unit and adding a histidine tag to the N-terminus to obtain a recombinant polypeptide unit; then connecting several recombinant polypeptide units in series from the N-terminus to the C-terminus to obtain the amino acid sequence of a recombinant protein; introducing the coding gene of the recombinant protein into Escherichia coli to construct a recombinant Escherichia coli; after inducing expression in the recombinant Escherichia coli, the wet bacteria are resuspended and crushed with a buffer solution to extract the recombinant protein; and the recombinant protein is treated with enterokinase to obtain a plurality of polypeptide units.

[0004] However, the prokaryotic expression system lacks protein modification function. The expressed proteins are not modified and may not have natural activity, so they are not suitable for producing eukaryotic exogenous proteins. In addition, Escherichia coli lacks transfer RNA corresponding to 7 rare codons of eukaryotic expression proteins. If the target protein contains a large number of amino acids translated by these codons, it will be difficult to express the full-length protein, or the expression level will be very low.

[0005] In summary, it is of great significance to develop a prokaryotic expression system that has certain protein modification functions and can efficiently express a variety of proteins. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a prokaryotic expression system and a preparation method and application thereof. The present invention genetically modifies the prokaryotic system, significantly enhances its protein modification function, expands the types of proteins that can be expressed, and increases the expression amount.

[0007] Compared with the prior art, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a prokaryotic expression system, comprising prokaryotic cells expressing prolyl hydroxylase functional subunits P4HB and P4HA1 and transfer RNA of codons AUA, AGG, AGA, CUA, CCC, GGA and CGG.

[0009] The present invention genetically modifies a prokaryotic system. By co-expressing prolyl hydroxylase functional subunits P4HB and P4HA1 in a prokaryotic expression system, P4HB and P4HA1 can perform hydroxylation modification on proline in collagen for recombinant collagen, that is, prokaryotic cells can be enabled to perform post-translational hydroxylation modification on proline on an amino acid chain, thereby expressing more accurate recombinant collagen. By co-expressing seven rare codons (AUA, AGG, AGA, CUA, CCC, GGA and CGG) in a prokaryotic expression system, prokaryotic cells can acquire the ability to translate amino acids corresponding to these codons, thereby increasing the expression amount of the protein, and having broad application prospects.

[0010] It is understood that any prokaryotic cells commonly used in the art for protein expression are suitable for the present invention.

[0011] Optionally, the prokaryotic cell includes Escherichia coli, and the Escherichia coli can be selected from the group consisting of, etc., without particular limitation.

[0012] It can be understood that the prolyl hydroxylase functional subunits P4HB and P4HA1, and transfer RNAs of codons AUA, AGG, AGA, CUA, CCC, GGA and CGG known in the art are all suitable for the prokaryotic expression system of the present invention without special limitation.

[0013] Preferably, the amino acid sequence of P4HB includes the sequence shown in SEQ ID NO.1.

[0014] Preferably, the amino acid sequence of P4HA1 includes the sequence shown in SEQ ID NO.2.

[0015] Preferably, the nucleic acid sequence of the transfer RNA of the codon AUA includes the sequence shown in SEQ ID NO.3.

[0016] Preferably, the nucleic acid sequence of the transfer RNA of the codon AGG includes the sequence shown in SEQ ID NO.4.

[0017] Preferably, the nucleic acid sequence of the transfer RNA of the codon AGA includes the sequence shown in SEQ ID NO.5.

[0018] Preferably, the nucleic acid sequence of the transfer RNA of the codon CUA includes the sequence shown in SEQ ID NO.6.

[0019] Preferably, the nucleic acid sequence of the transfer RNA of the codon CCC includes the sequence shown in SEQ ID NO.7.

[0020] Preferably, the nucleic acid sequence of the transfer RNA of the codon GGA includes the sequence shown in SEQ ID NO.8.

[0021] Preferably, the nucleic acid sequence of the transfer RNA of the codon CGG includes the sequence shown in SEQ ID NO.9.

[0022] SEQ ID NO.1:

[0023] VAKGFKGKALVVNVPHTEDRVLEFFGITKASLPAIVLVDMSGGSSMKKYPYDGASDDAAAITAHVSGVFEGKVKATLKSEEPSPADTAGDVVVLKGKSFNELV MDNEKDVLVEFYAPWCGHCKKLAPVYDELGAMYKDNENIVIAKMDSTANEIDVPGVDVKGFPTLYFFPGKDKKAVKYEGGREVEDFVDYLKKNAHNDAKHTEL.

[0024] SEQ ID NO.2:

[0025] MPFLIWLDDLEVSYMTGEDYMWATADWLDPQASMLATAADEQHMPSKEYNYRVAMEEPDIPQGFRFVGPTRAEAEAALPPQGQSPSTTFQARRFDEELQAYLEDNRVCNSSTCRTTPSAAGHKVGTLLRQHRGRNCSKARPRQSKLALHEAAEEICPEDN STQAKQPVAL LDLVEGALRQ.

[0026] SEQ ID NO.3:

[0027] gcatccatggctgaatggttaaagcgcccaactcataattggcgaattcgtaggttcaattcctactggatgca.

[0028] SEQ ID NO.4:

[0029] ttctttattagtttaatggtagaacaagatattcctaatatcctaatctaagttcgagtcttaggtaagaaa.

[0030] SEQ ID NO.5:

[0031] gcgtccatcgtctaatggataggacaggggtcttctaaacctccggtataggttcaaatcctattggacgta.

[0032] SEQ ID NO.6:

[0033] gccgccatggtgaaattggtagacacgctgctcttaggaagcagtgctagggcatctcggttcgaatccgagtggcggca.

[0034] SEQ ID NO.7:

[0035] cggagcatggcgcagcttggtagcgtgccatcttggggtgatggaggtcgcaggttcaaatcctgctgctccga.

[0036] SEQ ID NO.8:

[0037] tgctcttctagtatattaattacaattgacttccaatctctaaaatctggtgtaactccagagatgagcaa.

[0038] SEQ ID NO.9:

[0039] gggttggtagctcagtggatcagagaccatggtcccggaccatgaagtcaagggttcgaatccctcctagccca.

[0040] In a second aspect, the present invention provides a method for preparing the prokaryotic expression system according to the first aspect, the preparation method comprising:

[0041] The coding sequences of the functional subunits P4HB and P4HA1 of prolyl hydroxylase and the coding sequences of the transfer RNAs of the codons AUA, AGG, AGA, CUA, CCC, GGA and CGG are respectively inserted into expression vectors to obtain recombinant vectors, and the recombinant vectors are introduced into prokaryotic cells to obtain the prokaryotic expression system.

[0042] Preferably, the coding sequence of P4HB includes the sequence shown in SEQ ID NO.10.

[0043] Preferably, the coding sequence of P4HA1 includes the sequence shown in SEQ ID NO.11.

[0044] SEQ ID NO.10:

[0045] .

[0046] SEQ ID NO.11:

[0047] .

[0048] Preferably, the prokaryotic cell comprises Escherichia coli.

[0049] In a third aspect, the present invention provides use of the prokaryotic expression system described in the first aspect in producing proteins.

[0050] In a fourth aspect, the present invention provides a method for producing a protein, the method comprising:

[0051] The coding sequence of the target protein is inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is introduced into the prokaryotic expression system described in the first aspect, cultured and purified to obtain the target protein.

[0052] Optionally, the target protein includes collagen.

[0053] Optionally, the nucleotide sequence of the collagen includes the sequence shown in SEQ ID NO.12.

[0054] SEQ ID NO.12:

[0055] gggcctcaaggtattgctggacagcgtggtgtggtcggcctgcctggtcagagaggagagagaggcttccctggtcttcctggcccctctggtgaacctggcaaacaaggtccctctggagcaagtggtgaacgtggtccccctggtcccatgggcccccctggattggctggaccccctggtgaatctggacgtgagggggctcctggtgccgaaggttcccctggacgagacggttctcctggcgccaagggtgaccgtggtgagaccggccccgctggaccccctggtgctcctggtgctcctggtgcccctggccccgttggccctgctggcaagagtggtgatcgtggtgagactggtcctgctggtcccgccggagaacgaggtggccctggaggacctggccctcagggtcctcctggaaagaatggtgaaactggacctcagggacccccagggcctactgggcctggtggtgacaaaggagacacaggaccccctggtccacaaggattacaaggcttgcctggtacaggtggtcctccaggagaaaatggaaaacctggggaaccaggtccaaagggtgatgccggtgcacctggagctccaggaggcaagggtgatgctggtgcccctggtgaacgtggacctcctggattggcaggggccccaggacttagaggtggagctggtccccctggtcccgaaggaggaaagggtgctgctggtcctcctgggccacctggtgctgctggtactcctggtctgcaaggaatgcctggacctggtccttgctgtggtggttaa。

[0056] Preferably, the separation and purification method adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

[0057] Compared with the prior art

[0058] The present invention genetically modifies a prokaryotic system, and co-expresses prolyl hydroxylase functional subunits P4HB and P4HA1 in a prokaryotic expression system. P4HB and P4HA1 are targeted at recombinant collagen and can perform hydroxylation modification on proline in the collagen, that is, prokaryotic cells can be enabled to perform post-translational hydroxylation modification on proline on an amino acid chain, and more accurate recombinant collagen can be expressed; and seven rare codons (AUA, AGG, AGA, CUA, CCC, GGA and CGG) are co-expressed, so that prokaryotic cells can obtain the ability to translate amino acids corresponding to these codons, thereby increasing the expression amount of the protein, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the SDS-PAGE result diagram in Example 4. DETAILED DESCRIPTION

[0060] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0061] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0062] In the specific embodiment of the present invention, the production of recombinant collagen (nucleic acid sequence is shown as SEQ ID NO. 12) is taken as an example to verify the prokaryotic expression system of the present invention.

[0063] Example 1

[0064] In this example, a vector for co-expression of collagen, P4HB and P4HA1 was constructed.

[0065] The coding genes of collagen (SEQ ID NO.12), P4HB (SEQ ID NO.10) and P4HA1 (SEQ ID NO.11) were synthesized by whole gene synthesis and directly connected to the restriction sites NdeⅠ and BamHI of the expression vector pET28a in sequence to obtain the recombinant expression vector pET28a-COL-P4HB-P4HA1.

[0066] Example 2

[0067] In this example, expression vectors for transfer RNAs with codons AUA, AGG, AGA, CUA, CCC, GGA, and CGG were constructed.

[0068] The nucleic acid sequences SEQ ID NO.3 to SEQ ID NO.9 of the transfer RNA of AUA, AGG, AGA, CUA, CCC, GGA and CGG were synthesized by total gene synthesis and directly connected to the expression vector pUC57 in sequence to obtain the recombinant vector pUC57-tRNA.

[0069] The recombinant expression vector pGro7-tRNA was constructed by inserting each tRNA behind the arabinose promoter of the pGro7 vector.

[0070] (1) Primer design: The primers F1 and R1 for the amplification of the whole tRNA fragment were designed according to the primer design principles of RF cloning (unrestricted cloning).

[0071] (2) Using the synthetic plasmid containing the tRNA gene (pUC57-tRNA) as a template, the above-mentioned amplification primers were used to amplify the tRNA target gene, and the PCR product, i.e., the tRNA gene fragment (containing 7 tRNAs), was recovered using a DNA recovery and purification kit (TIANGEN).

[0072] The PCR amplification system is shown in Table 1.

[0073] Table 1

[0074] Reagents Dosage PrimeSTAR HS(premix) 25μL F1(10μM) 1μL R1(10μM) 1μL pUC57-tRNA 1μL (about 10ng) <![CDATA[ddH2O]]> 22μL

[0075] The PCR amplification program is shown in Table 2.

[0076] Table 2

[0077]

[0078] (3) RF linear amplification to construct vector pGro7-tRNA: pGro7 empty plasmid (TaKaRa) was used as a template and the above-mentioned recovered products were used as primers for the second round of PCR linear amplification. The PCR system is shown in Table 3.

[0079] Table 3

[0080]

[0081]

[0082] The reaction procedure is shown in Table 4.

[0083] Table 4

[0084]

[0085] After amplification, the DNA fragments were treated with DpnⅠ to digest a small amount of template plasmid in the reaction system. The enzyme digestion system is shown in Table 5. The conditions are: 37 degrees Celsius for 1 hour and 16 degrees Celsius overnight. The products after enzyme digestion were directly transformed into Escherichia coli DH5α strains according to the calcium chloride method. Positive transformants were picked for colony PCR verification and sent for sequencing comparison. The results were consistent, and the plasmid was extracted to obtain the recombinant expression plasmid pGro7-tRNA.

[0086] Table 5

[0087] Reagents Dosage (μL) 10×Buffer 2 Dpn Ⅰ enzyme 1 DNA 17

[0088] Example 3

[0089] This example constructs a prokaryotic expression system.

[0090] The recombinant expression plasmid pET28a-COL-P4HB-P4HA1 prepared in Example 1 and Example 2 and pGro7-tRNA were mixed at a molar ratio of 1:1, transformed into competent Escherichia coli BL21 (DE3) by the calcium chloride method, and coated on an LB plate containing kanamycin and chloramphenicol resistance to grow positive transformants. This is the final prokaryotic expression system BL21-COL-P4HB-P4HA1-tRNA capable of expressing collagen.

[0091] In addition, only pET28a-COL-P4HB-P4HA1 was used for transformation to obtain the prokaryotic expression system BL21-COL-P4HB-P4HA1, which was used as a subsequent control.

[0092] Example 4

[0093] This example uses the prokaryotic expression system constructed in Example 3 to produce recombinant collagen.

[0094] (1) Seed liquid preparation: Single colonies from the transformation plate in Example 3 were picked out on a clean bench and inoculated into 10 mL of LB liquid culture medium (Cm + and Kan + ) and cultured in a constant temperature shaker at 37°C and 200 rpm for 12 h as seed solution.

[0095] (2) Co-expression: The above seed solution was transferred to two shake flasks (Cm 200 mL / 1000 mL) at a volume ratio of 5:100. + and Kan +), BL21-COL-P4HB-P4HA1-tRNA was inoculated in No. 1, and pET28a-COL-P4HB-P4HA1 was inoculated in No. 2. Before inoculation, arabinose solution with a final concentration of 2 mg / mL was added to No. 1 flask to induce tRNA expression. After culturing at 37°C and 220 rpm in a constant temperature shaker for 2 h, the bacterial solution OD was measured. 600 When the value was 0.8, IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.1 mM was added to the No. 1 and No. 2 shake flasks to induce collagen expression and continue to culture for 5 hours. After the induction, the OD of the bacterial solution was measured. 600 The cells were centrifuged at 8000 rpm for 7 min at 4 °C to collect the cells. The supernatant was run on SDS-PAGE. Figure 1 As shown, bands appeared at about 24 KDa, and the band of BL21-COL-P4HB-P4HA1-tRNA was darker (lane 1), indicating a higher collagen content. This shows that the present invention co-expresses 7 rare codons (AUA, AGG, AGA, CUA, CCC, GGA and CGG), enabling prokaryotic cells to acquire the ability to translate the amino acids corresponding to these codons, thereby increasing the expression level of the protein.

[0096] PBS buffer was added at a ratio of 10OD / mL to completely suspend the bacteria, and the thoroughly suspended bacteria were placed in a high-pressure disruptor to disrupt the cells; after the disruption was completed, the supernatant and precipitate were obtained by centrifugation at 4°C and 8000rpm for 45min; the supernatant sample was collected, which was the crude protein expression solution, and the supernatant sample was filtered through a 0.22μm filter membrane to obtain a pre-column sample.

[0097] 30 mL of crude protein expression solution was adjusted to pH 6.0, and GE's capto S1 mL pre-packed column was used as the purification medium for crude purification. The crude purified sample was separated by molecular sieve. The amino acid composition of the purified collagen was analyzed. The protein concentration after ultrafiltration and concentration was determined using the BCA method and a protein concentration determination kit (Thermo Fisher). 10 mg of protein sample was added to a hydrolysis tube with 5 mL of 6 M hydrochloric acid, which was vacuumed and filled with nitrogen. The tube was placed in a 110 ° C oven for hydrolysis for 24 h. After hydrolysis, the hydrolysis tube was cooled to 25 ° C, and the volume was adjusted to 25 mL with ddH2O. 2 mL of nitrogen was taken and dried (add a small amount of ddH2O and dried twice), and finally 1 mL of pH 2.2, 0.02 M hydrochloric acid buffer was added to resuspend, filtered with a 0.45 μm filter membrane, and detected using a Hitachi L8900 amino acid automatic analyzer (Hitachi Co., Ltd., Japan). The results showed that the recombinant collagen contained hydroxyproline, indicating that the present invention successfully achieved the hydroxylation of the recombinant collagen by co-expressing the collagen gene and the proline hydroxylase gene.

[0098] In summary, the present invention genetically modifies the prokaryotic system, and co-expresses the functional subunits P4HB and P4HA1 of prolyl hydroxylase in the prokaryotic expression system. P4HB and P4HA1 are targeted at recombinant collagen and can perform hydroxylation modification on the proline in the collagen, that is, the prokaryotic cells can have the ability to perform post-translational hydroxylation modification on the proline on the amino acid chain, and express more accurate recombinant collagen; 7 rare codons (AUA, AGG, AGA, CUA, CCC, GGA and CGG) are co-expressed, so that the prokaryotic cells can obtain the ability to translate the amino acids corresponding to these codons, thereby increasing the expression amount of the protein, and having broad application prospects.

[0099] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A prokaryotic expression system, characterized in that: The prokaryotic expression system includes prokaryotic cells expressing collagen, prolyl hydroxylase functional subunits P4HB and P4HA1, and transfer RNA of codons AUA, AGG, AGA, CUA, CCC, GGA, and CGG; The prokaryotic cells include Escherichia coli; The amino acid sequence of P4HB is shown in SEQ ID NO.1; The amino acid sequence of P4HA1 is shown in SEQ ID NO.2; The nucleic acid sequence of the transfer RNA of the codon AUA is shown in SEQ ID NO.3; The nucleic acid sequence of the transfer RNA of the codon AGG is shown in SEQ ID NO.4; The nucleic acid sequence of the transfer RNA of the codon AGA is shown in SEQ ID NO.5; The nucleic acid sequence of the transfer RNA of the codon CUA is shown in SEQ ID NO.6; The nucleic acid sequence of the transfer RNA of the codon CCC is shown in SEQ ID NO.7; The nucleic acid sequence of the transfer RNA of the codon GGA is shown in SEQ ID NO.8; The nucleic acid sequence of the transfer RNA of the codon CGG is shown in SEQ ID NO.9; The nucleotide sequence of the collagen is shown in SEQ ID NO.

12.

2. A method for preparing the prokaryotic expression system according to claim 1, characterized in that: The preparation method comprises: The coding sequences of the functional subunits P4HB and P4HA1 of prolyl hydroxylase and the coding sequences of the transfer RNAs of the codons AUA, AGG, AGA, CUA, CCC, GGA and CGG are respectively inserted into expression vectors to obtain recombinant vectors, and the recombinant vectors are introduced into prokaryotic cells to obtain the prokaryotic expression system.

3. The method for preparing the prokaryotic expression system according to claim 2, characterized in that: The coding sequence of P4HB is shown in SEQ ID NO.10; The coding sequence of P4HA1 is shown in SEQ ID NO.

11.

4. The method for preparing the prokaryotic expression system according to claim 3, characterized in that: The prokaryotic cells include Escherichia coli.

5. Use of the prokaryotic expression system according to claim 1 in producing collagen.

6. A method for producing collagen, characterized in that: The method comprises: The prokaryotic expression system according to claim 1 is cultured and purified to obtain the protein.

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