Application of His-SUMO tag in preparation of recombinant HPV18 E7 protein

Abstract

The application discloses an application of a His-SUMO tag in preparation of a recombinant HPV18 E7 protein and belongs to the technical field of recombinant protein expression. The application is high-efficiency expression of a soluble HPV18 E7 fusion protein (His-SUMO-HPV18 E7) induced by a His+SUMO tag, and preparation of a recombinant HPV18 E7 protein (No tag HPV18 E7) without redundant tag amino acid sequences by affinity chromatography purification and tag removal technology, so as to solve the problems of low soluble expression amount and / or large molecular tag in the prior art. The prepared recombinant human papillomavirus HPV18 E7 protein has clear and single SDS-PAGE electrophoresis bands, good repeatability, high yield (up to 20.5 mg / L), and a protein purity of up to 99%, and can be effectively applied to large-scale production of the recombinant human papillomavirus HPV18 E7 protein.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of recombinant protein expression, and particularly relates to application of a His-SUMO tag in preparation of a recombinant HPV18 E7 protein. BACKGROUND

[0002] Recombinant protein refers to a protein obtained by using recombinant DNA or RNA technology. Generally, a target gene is first obtained by using gene cloning or chemical synthesis technology, connected to a suitable expression vector, introduced into a specific host cell, and a functional protein molecule is expressed by using the genetic system of the host cell and then subjected to protein purification. In the stage of constructing an expression vector, in addition to some necessary expression elements, codon optimization and selection of a tag also need to be considered.

[0003] In actual production, there are many types of tags, such as HIS, GST, MBP, NusA, SUMO and the like. The effects of different tags are not the same for different recombinant proteins. Therefore, finding a suitable protein tag has become a research focus. At the same time, a tag with a large molecular weight may shield the active site of the target protein, and has a certain immunogenicity. Exploring a set of tags suitable for the target protein and a tag removal technology can make the target protein achieve the best use effect.

[0004] Cervical cancer is a common female malignant tumor. Studies have shown that almost all cervical cancers are closely related to human papilloma virus (HPV). The E7 protein of high-risk HPV is continuously highly expressed in HPV-infected cells and plays an important role in the occurrence and development of cervical cancer, but the specific pathogenic mechanism is not clear.

[0005] At present, the expression amount of HPV18 E7 protein in Escherichia coli is low and the purification effect is not good, and most of them are expressed by using a GST tag for solubilization. Chen Xiaoli et al. expressed recombinantly by using a large GST fusion tag, the main band of the expression supernatant was not strong, and the large tag was not removed, and finally a small amount of HPV18 E7 recombinant protein with a GST tag was obtained. Lai Yongwei expressed recombinantly by using a GST fusion tag, and removed the tag by using prescission protease, and finally obtained 15 mg / L of HPV18 E7. However, the enzyme recognizes a short peptide Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro and cuts the enzyme between the Gln and Gly amino acid residues, and finally two amino acids are left on the target protein. The mass spectrometry identification result also shows that the first two amino acids at the N-terminus are Gly and Pro, and the author does not explain the final destination of the prescission protease. SUMMARY

[0006] 1.Object of the Invention

[0007] The object of the present application is to provide the application of His-SUMO tag in the preparation of recombinant HPV18 E7 protein, to induce the high-efficiency expression of soluble HPV18 E7 fusion protein (His-SUMO-HPV18 E7) by His+SUMO tag, and to prepare recombinant HPV18 E7 protein (Notag HPV18 E7) without extra tag amino acid sequence by affinity chromatography purification and tag removal technology, so as to solve the problems of low soluble expression and / or large molecular tag in the prior art.

[0008] 2.Technical Solution

[0009] In order to achieve the above object of the application, the technical solution adopted by the present application is as follows:

[0010] The present application provides the application of His-SUMO tag in the preparation of recombinant HPV18 E7 protein.

[0011] Further, the amino acid sequence of the above-mentioned recombinant HPV18 E7 protein is shown in SEQ ID NO. 1.

[0012] Further, the amino acid sequence of the above-mentioned His-SUMO tag is shown in SEQ ID NO. 4.

[0013] Further, the above-mentioned application comprises the following steps:

[0014] S1, fusion plasmid construction, the fusion plasmid is an expression vector containing a polynucleotide sequence encoding a fusion protein His-SUMO-HPV18 E7;

[0015] S2, fusion protein expression, transforming the fusion plasmid in S1 into E. coli and inducing expression, collecting E. coli bacteria, breaking the bacteria, centrifuging to collect the supernatant, and the supernatant containing the fusion protein His-SUMO-HPV18 E7;

[0016] S3, fusion protein purification, purifying the supernatant in S2 by Ni-Smart agarose gel affinity chromatography to obtain high-purity fusion protein His-SUMO-HPV18 E7;

[0017] S4, fusion protein enzyme cutting, using SUMO enzyme for enzyme cutting to obtain an enzyme cutting mixture;

[0018] S5, recombinant protein purification, purifying the enzyme cutting mixture in S4 by Ni-Smart agarose gel affinity chromatography to obtain high-purity recombinant HPV18 E7 protein without tag.

[0019] Further, in the S1 fusion plasmid construction, the polynucleotide sequence encoding the recombinant HPV18 E7 protein is shown as SEQ ID NO. 5.

[0020] Further, in the S1 fusion plasmid construction, the polynucleotide sequence encoding the His-SUMO tag is shown as SEQ ID NO. 8.

[0021] Further, in the S1 fusion plasmid construction, the polynucleotide sequence encoding the fusion protein His-SUMO-HPV18 E7 is shown as SEQ ID NO. 7.

[0022] Further, in the S1 fusion plasmid construction, the expression vector includes pET-28a.

[0023] Further, the S2 fusion protein expression specifically includes: transforming E. coli with the fusion plasmid in S1, inoculating, expanding culture, inducing expression, collecting bacterial cells, breaking bacterial cells, and centrifuging to collect supernatant.

[0024] Further, the S3 fusion protein purification specifically includes: equilibrating the Ni-Smart chromatography column with PBS buffer to a baseline smoothness; loading at a flow rate of 0.5 mL / min; after loading is completed, washing the chromatography column with PBS containing 0.5M NaCl and 25mM imidazole to a baseline smoothness; eluting the target protein with PBS containing 0.5M NaCl and 500mM imidazole; replacing with PBS buffer by ultrafiltration to remove 0.5M NaCl and 500mM imidazole, obtaining high-purity fusion protein His-SUMO-HPV18 E7.

[0025] Further, the S4 fusion protein digestion specifically includes: a mass ratio of fusion protein to SUMO enzyme of 20:1, and enzyme digestion at 30°C for 1h, obtaining an enzyme digestion mixture containing recombinant HPV18 E7 protein without a tag, cut-off His-tagged SUMO tag, and His-tagged SUMO enzyme.

[0026] Further, the S5 recombinant protein purification specifically includes: equilibrating the Ni-Smart chromatography column with PBS buffer to a baseline smoothness; loading the enzyme digestion mixture at a flow rate of 0.1 mL / min; collecting the flow-through liquid to obtain high-purity recombinant human papillomavirus HPV18 E7 protein without a tag.

[0027] The application also provides a production method of recombinant HPV18 E7 protein, specifically including:

[0028] S1, fusion plasmid construction, the fusion plasmid is an expression vector containing a polynucleotide sequence encoding a fusion protein His-SUMO-HPV18 E7;

[0029] S2, fusion protein expression, transforming the fusion plasmid in S1 into E. coli and inducing expression, collecting E. coli bacteria, breaking the bacteria, collecting the supernatant by centrifugation, and the supernatant containing the fusion protein;

[0030] S3, fusion protein purification, purifying the supernatant in S2 by Ni-Smart agarose gel affinity chromatography to obtain high-purity fusion protein;

[0031] S4, fusion protein digestion, using SUMO enzyme for digestion to obtain a digestion mixture;

[0032] S5, recombinant protein purification, purifying the digestion mixture in S4 by Ni-Smart agarose gel affinity chromatography to obtain high-purity recombinant HPV18 E7 protein without tag.

[0033] 3. Beneficial effects

[0034] Compared with the prior art, the application has the beneficial effects that:

[0035] (1) The application provides the application of His-SUMO tag in the preparation of recombinant HPV18 E7 protein. Through the His+SUMO tag expression system and the enzyme digestion technology, the soluble recombinant human papillomavirus HPV18 E7 protein without tag can be prepared. The recombinant human papillomavirus HPV18 E7 protein prepared has clear and single SDS-PAGE electrophoresis bands, good repeatability, high yield (up to 20.5 mg / L), and protein purity up to 99%, which can be effectively applied to large-scale production of recombinant human papillomavirus HPV18 E7 protein.

[0036] (2) The application provides the application of His-SUMO tag in the preparation of recombinant HPV18 E7 protein. Through genetic engineering technology, an expression vector is constructed in vitro, the high-efficiency expression of soluble HPV18 E7 fusion protein (His-SUMO-HPV18 E7) is induced by His+SUMO tag, and the recombinant HPV18 E7 protein (No tag HPV18 E7) without redundant tag amino acid sequences is prepared by affinity chromatography purification and tag removal technology. The purity reaches 99%, 1L of culture solution finally obtains 20.5 mg of target protein, the purity and yield meet the industrial demand, solve the problems of low soluble expression amount and / or containing macromolecular tag of HPV18 E7 in the prior art, and lay a foundation for further development of HPV in vitro detection reagents and HPV therapeutic vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a schematic diagram for plasmid construction.

[0038] Figure 2 Figure 2 is a plasmid map of pET-28a.

[0039] Figure 3 Figure 3 is a SDS-PAGE electrophoretogram of expression and purification of His- SUMO-HPV18 E7 fusion protein.

[0040] Figure 4 Figure 4 is a SDS-PAGE electrophoretogram of expression and purification of His- HPV18 E7 fusion protein.

[0041] Figure 5 Figure 5 is a SDS-PAGE electrophoretogram of enzyme digestion and recovery of HPV18 E7 fusion protein.

[0042] Figure 6 Figure 6 is a supernatant precipitation diagram of CXCL-13 protein expression with different tags.

[0043] Figure 7 Figure 7 is a supernatant precipitation diagram of MiTF protein expression with different tags.

[0044] Figure 8 Figure 8 is a supernatant precipitation diagram of HPV18 E6 protein expression with different tags.

[0045] Figure 9 Figure 9 is a supernatant precipitation diagram of HPV16 E6 protein expression with different tags. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with specific examples.

[0047] It should be noted that the terms such as "upper", "lower", "left", "right", "intermediate" and the like cited in the present specification are merely for the convenience of clear description, and are not intended to limit the scope of the application. Changes or adjustments of the relative relationship, without substantial changes in the technical content, are also regarded as the scope of the application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specified by the manufacturer, are all conventional products that can be purchased on the market.

[0050] As used herein, the term "about" is used to provide flexibility to a given term, measurement, or value associated with a term, measurement, or value. One of ordinary skill in the art can readily determine the degree of flexibility of a particular variable.

[0051] As used herein, the term "at least one of' is intended to be synonymous with "one or more of As an example, "at least one of A, B, and C" explicitly includes just A, just B, just C, and any combination thereof.

[0052] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to drop out any element not specifically recited. For example, description should be read to include not only the generic description of a numerical range, but also to the inclusion of any numerical whole number within that range, as well as to the inclusion of any sub-range within that range. It is to be understood that such a statement is merely a statement of application and is not a limitation on the scope of the application. This same principle applies to ranges reciting only one numerical value as minimum and / or maximum.

[0053] Example 1

[0054] This example provides the application of His-SUMO tag in the preparation of recombinant HPV18 E7 protein. Specifically includes the following steps:

[0055] S1, construction of fusion plasmid for expressing fusion protein

[0056] The amino acid sequence of HPV18 E7 protein in this example is shown as SEQ ID NO. 1, and the polynucleotide sequence (gene sequence) encoding the protein is shown as SEQ ID NO. 5; the amino acid sequence of His-SUMO tag is shown as SEQ ID NO. 4, and the polynucleotide sequence (gene sequence) encoding His-SUMO tag is shown as SEQ ID NO. 8; the His-SUMO-HPV18 E7 fusion protein is constructed by adding histidine tag (8xHis) and SUMO tag (His-SUMO tag) at the N terminus of the target protein (HPV18 E7 protein), the amino acid sequence of His-SUMO-HPV18 E7 fusion protein is shown as SEQ ID NO. 3, and the polynucleotide sequence (gene sequence) encoding His-SUMO-HPV18 E7 fusion protein is shown as SEQ ID NO. 7. The specific construction method is shown in Figure 1 The plasmid pET-28a (provided by Suzhou Hongxun, as shown in Figure 2The amino acid sequence of the His-HPV18 E7 fusion protein is shown as SEQ ID NO. 2, and the polynucleotide sequence (gene sequence) encoding the His-HPV18 E7 fusion protein is shown as SEQ ID NO. 6. The construction method is the same as that of the fusion plasmid pET-28a-His-SUMO-HPV18 E7, and the fusion plasmid pET-28a-His-HPV18 E7 expressing the His-HPV18 E7 fusion protein is obtained. The polynucleotide sequences in this embodiment were synthesized by Suzhou Hongxun.

[0057] Table 1 Amino acid and polynucleotide sequences

[0058]

[0059]

[0060] S2, fusion protein recombinant expression and preparation of purified supernatant

[0061] Take 0.5 μg of the fusion plasmid pET-28a-His-SUMO-HPV18 E7 and pET-28a-His-HPV18 E7 in S1, respectively, and transform into E. coli BL21 competent cells (Weidi Biology, Catalog No: EC1002M), add 1 mL of LB medium (without resistance), 37°C, 200 rpm shaking bacteria for 1 h. Centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, resuspend the bacteria with the remaining culture medium, and spread on LB plates containing 100 μg / mL kanamycin (Shanghai Shengao, Catalog No: A600286), pick single colonies and inoculate in 50 mL of LB medium containing 100 μg / mL kanamycin, 37°C, 200 rpm shaking bacteria for 16 h.

[0062] Inoculate 2% (v / v) into 200 mL of fresh LB liquid medium containing 100 μg / mL kanamycin, and incubate at 37°C until the bacterial concentration reaches OD 600To about 0.6, IPTG (Shanghai Shenguo, item number: A600168) was added to the LB liquid medium to a final concentration of 1 mM, and the culture was induced at 20°C for 16 h, then centrifuged at 12000 rpm for 10 min at 4°C, and the wet bacteria were collected. Resuspend with 200 mM PBS buffer at pH 7.4, then perform ultrasonic crushing (ultrasonic power is 250 W, ultrasonic 2 s, interval 6 s, effective ultrasonic time 10 min), and the crushed suspension was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was taken to obtain two crude enzyme solutions of the fusion proteins His-SUMO-HPV18 E7 and His-HPV18 E7.

[0063] S3, purification of the fusion protein

[0064] The target protein was separated and purified by His tag affinity chromatography, and the filler used was Ni-Smart (Changzhou Tian Dihuan, item number: SA036500). A 5 mL Ni-Smart chromatography column was equilibrated to the baseline with 200 mM PBS buffer at pH 7.4. The two crude enzyme solutions of the fusion proteins His-SUMO-HPV18 E7 and His-HPV18 E7 in S2 were filtered with a 0.45 μm filter (Merck Millipore, item number: SLHPR33RB) at a flow rate of 0.5 mL / min, and then loaded onto the 5 mL Ni Smart chromatography column previously equilibrated with PBS. After loading, the chromatography column was washed with PBS containing 0.5 M NaCl and 25 mM imidazole until the baseline was stable, and finally the target protein was eluted with PBS containing 0.5 M NaCl and 500 mM imidazole; ultrafiltration was used to replace the PBS buffer to remove 0.5 M NaCl and 500 mM imidazole, and finally 18.6 mg of His-SUMO-HPV18 E7 fusion protein was obtained, with a yield of 93 mg / L; and 1.4 mg of His-HPV18 E7 fusion protein was obtained, with a yield of 7 mg / L. Small samples were taken for SDS-PAGE analysis, and the results are shown in Figure 3 and Figure 4 As shown, the His-SUMO-HPV18 E7 fusion protein has high purity, while the His-HPV18 E7 fusion protein not only has low yield, but also does not meet the industrial requirements in terms of purity.

[0065] S4, tag removal of the fusion protein

[0066] His-SUMO-HPV18 E7 fusion protein and SUMO enzyme were added at a mass ratio of 20:1, and SUMO enzyme (Sigma, item number: SAE0067, with His tag) was added. 18.6 mg of His-SUMO-HPV18 E7 fusion protein in S3 was diluted with PBS to a concentration of 1 mg / mL, and 0.93 mg of SUMO enzyme was added. The enzyme was cut at 30°C for 1 h to obtain an enzyme cutting mixed solution containing HPV18 E7 recombinant protein without a tag, cut His-tagged SUMO tag (His-SUMO), and His-tagged SUMO enzyme. Take a small sample of the enzyme cutting mixture at 0 h and 1 h for gel running.

[0067] S5, HPV18 E7 recombinant protein recovery

[0068] The Ni-Smart chromatography column was equilibrated with PBS buffer to baseline stability; the enzyme cutting mixture for 1 h was loaded at a flow rate of 0.1 mL / min; the flow-through was collected, and a small sample was taken together with the 0 h and 1 h enzyme cutting mixture samples in S4 for SDS-PAGE analysis, and the results are shown in Figure 5 The reaction started immediately after the addition of SUMO enzyme, and the fusion protein was completely cut after 1 h of reaction. We recovered 4.1 mg of recombinant human papillomavirus HPV18 E7 protein without a tag (No tag HPV18 E7) with a purity of more than 99% (Image J grayscale analysis), and the final yield was 20.5 mg / L.

[0069] Comparative example

[0070] This comparative example provides the application of His-SUMO tag in the preparation of other recombinant proteins. Refer to Example 1, which specifically includes:

[0071] Four other groups of fusion protein plasmids were constructed according to S1 of Example 1:

[0072] pET-28a-His-CXCL-13, pET-28a-His-SUMO-CXCL-13;

[0073] pET-28a-His-MiTF, pET-28a-His-SUMO-MiTF;

[0074] pET-28a-His-HPV18 E6, pET-28a-His-SUMO-HPV18 E6;

[0075] pET-28a-His-HPV16 E6, pET-28a-His-SUMO-HPV16 E6;

[0076] The amino acid sequence and gene sequence of the protein thereof are shown in SEQ ID NO. 9-SEQ ID NO. 32 (Table 2).

[0077] The four groups of fusion protein plasmids were induced for expression, broken wall operation, and the supernatant was precipitated and run on gel for verification. Figure 6 It is shown that the CXCL-13 protein, His-tagged and His-SUMO-tagged are not expressed; Figure 7 、 Figure 8 、 Figure 9 It is shown that the MiTF protein, HPV18 E6 protein and HPV16 E6 protein, His-tagged and His-SUMO-tagged are inclusion body expression, indicating that His-SUMO tag does not have the effect of soluble expression on the above four proteins, that is, His-SUMO tag has different effects on different proteins, and whether it can work or not, its effect is unknown.

[0078] Table 2 Amino acid and gene sequence of the comparative example

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] In summary, the present application found that the His+SUMO tag expression system and enzyme cutting technology can prepare the soluble recombinant human papillomavirus HPV18 E7 protein without tag, and the SDS-PAGE electrophoresis band is clear and single, the repeatability is good, the yield is high (up to 20.5 mg / L), which can be effectively applied to the large-scale production of recombinant human papillomavirus HPV18 E7 protein.

Claims

1. The application of the His-SUMO tag in the preparation of recombinant HPV18 E7 protein, characterized in that, The amino acid sequence of the recombinant HPV18 E7 protein is shown in SEQ ID NO.1; The amino acid sequence of the His-SUMO tag is shown in SEQ ID NO.4; the application includes: S1, Construction of fusion plasmid, wherein the fusion plasmid is an expression vector containing a multinucleotide sequence encoding the fusion protein His-SUMO-HPV18 E7; S2, fusion protein expression: The fusion plasmid in S1 was transformed into E. coli and induced to express. E. coli cells were collected, the cells were broken, and the supernatant was collected by centrifugation. The supernatant contained the fusion protein His-SUMO-HPV18 E7. S3, fusion protein purification: The supernatant in S2 was purified by Ni-Smart agarose gel affinity chromatography to obtain the fusion protein His-SUMO-HPV18 E7. S4, fused protein digestion, and then digested with SUMO enzyme to obtain a digestion mixture; S5, recombinant protein purification: The enzyme digestion mixture in S4 was purified by Ni-Smart agarose gel affinity chromatography to obtain untagged recombinant HPV18 E7 protein.

2. The application according to claim 1, characterized in that, The polynucleotide sequence encoding the fusion protein His-SUMO-HPV18 E7 is shown in SEQ ID NO.

7.

3. The application according to claim 2, characterized in that, The expression of the S2 fusion protein specifically includes: transforming the fusion plasmid in S1 into Escherichia coli, inoculating, expanding culture, inducing expression, collecting E. coli cells, lysing the cells, and centrifuging to collect the supernatant.

4. The application according to claim 2 or 3, characterized in that, The purification of the S3 fusion protein specifically includes: equilibrating the Ni-Smart chromatography column with PBS buffer until the baseline is stable; loading the sample at a flow rate of 0.5 mL / min; after loading, washing the chromatography column with PBS containing 0.5 M NaCl and 25 mM imidazole until the baseline is stable; eluting the target protein with PBS containing 0.5 M NaCl and 500 mM imidazole; ultrafiltration to replace the PBS buffer to remove 0.5 M NaCl and 500 mM imidazole, yielding the fusion protein His-SUMO-HPV18E7.

5. The application according to claim 4, characterized in that, The S4 fusion protein digestion specifically includes: a mass ratio of fusion protein to SUMOase of 20:1, digestion at 30°C for 1 h to obtain a digestion mixture.

6. The application according to claim 5, characterized in that, The purification of the S5 recombinant protein specifically includes: equilibrating the Ni-Smart chromatography column with PBS buffer until the baseline is stable; loading the enzyme digestion mixture at a flow rate of 0.1 mL / min; collecting the flow-through to obtain untagged recombinant HPV18 E7 protein.

7. A method for producing recombinant HPV18 E7 protein, characterized in that, The method includes: S1, Construction of fusion plasmid, wherein the fusion plasmid is an expression vector containing a multinucleotide sequence encoding the fusion protein His-SUMO-HPV18 E7; S2, fusion protein expression: The fusion plasmid in S1 is transformed into E. coli and induced to express. E. coli cells are collected, the cells are broken, and the supernatant is collected by centrifugation. The supernatant contains the fusion protein. S3, fusion protein purification: The supernatant in S2 was purified by Ni-Smart agarose gel affinity chromatography to obtain the fusion protein; S4, fused protein digestion, and then digested with SUMO enzyme to obtain a digestion mixture; S5, recombinant protein purification: The enzyme digestion mixture in S4 was purified by Ni-Smart agarose gel affinity chromatography to obtain untagged recombinant HPV18 E7 protein. The polynucleotide sequence encoding the fusion protein His-SUMO-HPV18 E7 is shown in SEQ ID NO.7.

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