CN110777148B

CN110777148B - DNA sequences for expression of FGF-2 and methods for producing FGF-2

Info

Publication number: CN110777148B
Application number: CN201910543546.4A
Authority: CN
Inventors: 钟树根; 邝纬阳
Original assignee: Mengqian Technology Intellectual Property Co ltd
Current assignee: Mengqian Technology Intellectual Property Co ltd
Priority date: 2019-06-21
Filing date: 2019-06-21
Publication date: 2021-08-06
Anticipated expiration: 2039-06-21
Also published as: CN110777148A; WO2020253287A1

Abstract

The invention provides a DNA sequence for expressing FGF-2, wherein the sequence is shown in SEQ ID NO.1 or SEQ ID NO.2 or a complementary sequence thereof. The invention also provides a construct, wherein the construct comprises a DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2 between EcoRI and NotI restriction sites of the plasmid vector or a complementary sequence thereof. The invention also provides a preparation method and application of the construct. In addition, the invention also provides a method for preparing FGF-2, wherein the method comprises the following steps: a. culturing the cells: culturing a mammalian cell; b. transfection: transfecting the construct into the mammalian cells obtained in step a; c. and (3) purification: FGF-2 was purified by affinity chromatography. The DNA sequence for expressing FGF-2 and the construction thereof provided by the invention can obviously improve the expression of FGF-2 with biological activity, thereby greatly reducing the cost for preparing and purifying FGF-2.

Description

DNA sequences for expression of FGF-2 and methods for producing FGF-2

Technical Field

The invention relates to a DNA sequence for expressing FGF-2, a construct containing the DNA sequence, a preparation method of the construct, application of the construct in expressing FGF-2, and a method for preparing FGF-2.

Background

FGF2 is a very valuable protein in the pharmaceutical and health care industries. FGF2 is an effective therapeutic protein for the treatment of neurodegenerative diseases, heart disease, angiogenesis, difficult-to-heal wounds and bone fractures; it also plays an important role in the large-scale production of stem cells.

Coli has long been used for purification of recombinant proteins [1-4] due to low production cost, high replication rate, and high productivity. However, the use of prokaryotic expression systems often encounters obstacles in the purification of mammalian proteins due to toxicity caused by foreign proteins, lack of correct protein folding and post-translational modification [5,6 ]. Scientists have used eukaryotic hosts such as yeast and insect cells to overcome these limitations for many years [7 ]. Nevertheless, the use of cultured cells of human origin for human-derived protein expression appears to be intuitive. Indeed, an increasing trend has arisen towards the production of recombinant proteins using mammalian cells [8,9 ].

Currently, one of the most widely used host systems for FGF2 production and purification is bacteria. However, simple prokaryotes lack the necessary post-translational modifications, including splicing, glycosylation and disulfide bonding, activity and solubility of the protein for purification. The natural folding of the purified protein is critical to its function and solubility. Protein aggregates or inclusion bodies are often found when eukaryotic proteins are expressed in bacterial systems. Methods involving inducing proteins or denaturing and renaturing protein aggregates at lower temperatures do not always produce good yields. Therefore, the preparation and purification of biologically active FGF2 is extremely expensive ($ 4000/mg), which prevents its widespread use.

Fusion of affinity tags such as polyhistidine (His-tag), glutathione S-transferase (GST) or Calmodulin Binding Peptide (CBP) to target proteins is a common strategy for protein purification [10 ]. The tag provides a simple, simplified purification scheme for crude cell lysates. Although the tag may be removed after purification, some additional amino acids are usually left on the cleaved protein derivative. These additional amino acids, although rare, can alter the folding and thus alter its native form [11-14 ]. Recombinant proteins with additional and unwanted amino acids may lead to irreversible deleterious effects, bind off-target receptors, and may lead to cancer [15,16] and allergy [16,17 ]. Recently, many groups have reported the use of protein introns or inteins to facilitate the purification of unlabeled proteins [18-21 ]. Inteins are self-cleaving elements in naturally occurring proteins. The first identified intein was found, the VMA gene encoding vascular ATPase, where the intein was cleaved and then the N-and C-terminal exteins were ligated to produce a functional full-length protein [19-24 ]. Now that over 400 inteins have been found in many organisms, scientists have taken advantage of this self-excision property in the field of protein purification. Site-specific, precise cleavage of inteins can produce human-identical protein products when designed appropriately [23,25 ].

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown as SEQ ID NO. 1:

CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAG GACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGG ATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTAC GGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGA AGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGG TACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTG CGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACA ATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGT TGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACA GGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAA GTCTTAATAATAA, or the complement thereof.

The invention also provides another DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown as SEQ ID NO. 2:

CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCC TGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGA GAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGC AACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCG AGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGAT CAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAG ATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGA TGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGA GATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATC GTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACA ACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAG GGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGC CTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACG GCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGA CCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAG GATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATC CGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATC CATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATC CGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGT TAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAA GAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGT GCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTAT AGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGA CCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAA GGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA, or the complement thereof.

In another aspect, the invention provides a construct comprising the DNA sequence shown in SEQ ID No.1 or the complement thereof between the EcoRI and NotI restriction sites of the plasmid vector or the DNA sequence shown in SEQ ID No.2 or the complement thereof between the EcoRI and NotI restriction sites of the plasmid vector.

In another aspect, the present invention provides a method for preparing a construct, comprising the steps of:

a. synthesizing a DNA sequence shown in SEQ ID NO.1 containing restriction sites of EcoRI and NotI or a complementary sequence thereof; or synthesizing a DNA sequence shown in SEQ ID NO.2 containing EcoRI and NotI restriction enzyme cutting sites or a complementary sequence thereof;

b. digesting the plasmid vector with EcoRI and NotI, and recovering the large fragment;

c. and (c) connecting the DNA sequence obtained in the step a or a complementary sequence thereof with the large fragment obtained in the step b by using ligase.

d. And c, transfecting the cell with the ligation product obtained in the step c, screening positive clones, and extracting plasmids.

In another aspect, the invention provides the use of a construct provided herein for the expression of FGF-2.

In another aspect, the present invention provides a method for preparing FGF-2, the method comprising:

a. culturing the cells: culturing a mammalian cell;

b. transfection: transfecting the mammalian cells obtained in step a with the construct as described above;

c. and (3) purification: FGF-2 was purified by affinity chromatography.

The DNA sequence and the construction body thereof for expressing FGF-2 provided by the invention can obviously improve the expression level of FGF-2, and the expressed FGF-2 is basically soluble, and the biological activity thereof is equivalent to that of the commercially available FGF-2. Therefore, by adopting the construct provided by the invention to express FGF-2, the expression of FGF-2 with biological activity can be obviously improved, and the cost for preparing and purifying FGF-2 can be greatly reduced.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention.

FIG. 1 is a schematic representation of the FGF2 construct and the his-DnaE-FGF2 construct. (A) DNA for the mature functional fragment of the FGF2 gene was synthesized and cloned into the pcDNA3.1(+) vector under the control of the CMV enhancer/promoter to form the pcDNA3.1-FGF2 construct. (B1) Synthesizing NF-kB binding sites and CREB binding sites, and cloning the NF-kB binding sites and the CREB binding sites behind a CMV promoter to form a pNC1 vector; (B2) the DNA of the mature functional fragment of the FGF2 gene was then cloned into pNC1 vector to form pNC1-FGF2 construct. (C) The 6xhis tag and Npu DnaE intein gene were fused to FGF2 by fusion PCR and cloned into pNC1 vector to form pNC1-6xhis-DnaE-FGF2 construct.

FIG. 2 shows the expression of the mature functional fragment FGF2 protein in HEK293T cells. (A) pcDNA3.1-FGF2 and pNC1-FGF2 constructs were transfected into HEK293T cells and whole cell lysates were analyzed by Western blotting at the indicated time points. (B) Densitometric analysis normalized to (a) relative β -actin. The pNC1-FGF2 construct showed higher expression levels at 24 hours, 36 hours and 48 hours (p <0.01, p < 0.001; n ═ 5 at each time point) than the pcdna3.1-FGF2 construct. (C) Supernatants (S) and pellets (P) from cell lysates were analyzed by western blot. FGF2(+ ve) was purchased commercially as a positive control. For both pcDNA3.1-FGF2 and pNC1-FGF2 constructs, FGF2 expressed by HEK293T was quite soluble.

Fig. 3 is a purification of FGF2 expressed in HEK293T cells. (A) Whole cell lysates and eluates from size exclusion chromatography samples were subjected to SDS-PAGE and stained with coomassie brilliant blue. (B) Samples from (a) were lyophilized and reconstituted in 0.1x PBS and then analyzed by silver staining after SDS-PAGE. Only 1 weak band (indicated by arrow) was observed in purified FGF 2.

Fig. 4 is a bioassay of purified FGF 2. (A) MTT assay to measure the effect of FGF2 on cell viability of C2C12 cells relative to PBS control. At the indicated time points, it was observed that 1ng/mL of commercial and purified FGF2 was able to induce cell growth in the culture medium (n-3). (B) Commercial and purified FGF2 was added to the medium at a concentration of 2ng/mL for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) was observed in PC12 cells 3 days after FGF2 treatment.

FIG. 5 shows the expression of the mature functional fragment FGF2 protein in HEK293T cells and the purification of FGF2 by intein-assisted cleavage. The pNC1-6xhis-DnaE-FGF2 construct was transfected into HEK293T cells, the medium was collected and spun at 2,000g for 10min to remove cell debris, and filtered through a 0.45 μm filter. The protein was purified by Ni-NTA affinity chromatography. Cleavage of the DnaE intein was induced in cleavage buffer. Starting 2 hours after induction, the full-length his-DnaE-FGF2 protein (band indicated by x) was cleaved into FGF2 (band indicated by #). Lysis was complete after 6 hours post-induction.

Figure 6 is a bioassay of purified and endoproteolytically cleaved FGF 2. (A) MTT assay to measure the effect of FGF2 on cell viability of C2C12 cells relative to PBS control. Both purified and endoproteolytic FGF2 were observed to be able to induce cell growth at the indicated time points at 1ng/mL in the culture medium (n-3). (B) 2ng/mL of purified and intein-cleaved FGF2 was added to the medium for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) was observed in PC12 cells 3 days after FGF2 treatment.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

The invention provides a DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown in SEQ ID NO. 1:

The invention also provides a DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown in SEQ ID NO. 2:

CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCC TGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGA GAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGC AACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCG AGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGAT CAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAG ATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGA TGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGA GATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATC GTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACA ACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAG GGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGC CTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACG GCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGA CCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAG GATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATC CGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATC CATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATC CGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGT TAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAA GAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGT GCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTAT AGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGA CCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAA GGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA, or the complement thereof. The DNA sequence fuses the Npu DnaE intein gene to facilitate intein-mediated separation and purification.

The invention also provides a construct which comprises the DNA sequence shown in SEQ ID No.1 between the EcoRI and NotI restriction sites of the plasmid vector or is the complementary sequence thereof, or the construct which comprises the DNA sequence shown in SEQ ID No.2 between the EcoRI and NotI restriction sites of the plasmid vector or is the complementary sequence thereof.

For the construct of the present invention, although there is no particular requirement for a plasmid, in order to increase the expression level of FGF-2, in a preferred embodiment, the plasmid vector is pcDNA3.1(+) or pNC1, more preferably pNC 1.

In order to further increase the expression level of FGF-2 and to facilitate purification, in a preferred embodiment the construct comprises the DNA sequence shown in SEQ ID NO.2 or the complement thereof between the EcoRI and NotI restriction sites of a plasmid vector, pNC 1.

The invention also provides a preparation method of the construct, which is characterized by comprising the following steps:

In a preferred embodiment, the plasmid vector is pcDNA3.1(+) or pNC1, more preferably pNC 1.

The invention also provides the use of the construct in the expression of FGF-2.

The invention also provides a method for preparing FGF-2, which is characterized by comprising the following steps:

a. culturing the cells: culturing a mammalian cell;

c. and (3) purification: FGF-2 was purified by affinity chromatography.

In a preferred embodiment, the affinity chromatography is performed by a heparin-binding domain.

In a preferred embodiment, in said step b, the mammalian cells obtained in step a are transfected with a construct as described above; the affinity chromatography is mediated by the intein DnaE.

In the method for producing FGF-2 of the present invention, although there is no particular limitation on mammalian cells, for example, HEK293T, HeLa, C2C12, etc. can be used. In a preferred embodiment, however, the mammalian cell is HEK 293T.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Examples

Materials and methods

Chemicals, plasmids and antibodies, all chemicals were purchased from Sigma-Aldrich (st. louis, MO), unless otherwise specified.

Construction of plasmid vector pNC1

As shown in B1 in fig. 1:

a. PCR (using GeneArt Strings Gene Synthesis service (Thermo-Fisher Scientific, Waltham, Mass.)) was used to synthesize an enhancer sequence, which is a DNA sequence containing the DNA sequence of the NF-. kappa.B binding site and the DNA sequence of the CREB binding site;

b. plasmid vector pcDNA was digested with NheI and HindIII 3.1 (+);

c. digesting the DNA sequence synthesized in the step a by using NheI and HindIII;

d. ligating the digested plasmid vector pcDNA3.1(+) obtained in step b with the digested DNA sequence obtained in step c using a ligase.

The sequence was confirmed by Sanger sequencing.

The specific process is as follows:

material

pcDNA3.1(+) was purchased from Thermo Scientific, USA, and its vector map is shown in FIG. 1A. Restriction enzymes NheI and HindIII, and T4DNA Ligase were purchased from NEB. Plasmid extraction kit and DNA fragment recovery kit were purchased from Thermo Scientific.

Enhancer PCR amplification

The enhancer template is synthesized by GeneArt string service (thermos scientific), and the DNA sequence of the enhancer is shown in SEQ ID NO. 5: 5'

GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTC AAC are provided. The forward and reverse primers used are shown in Table 1. The following reactants were added to the EP tube in order: mu.l of template, 1 Xbuffer, 1. mu.M forward primer, 1. mu.M reverse primer, 0.5. mu.l polymerase, 4. mu.l dNTP, and ddH2O to 50. mu.l of reaction system.

Reaction conditions are as follows: 95 deg.C for 5 min; 95 ℃ for 15 s; 55 ℃ for 10 s; 72 ℃ for 10 s; 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃.

And (3) carrying out electrophoresis on the PCR amplification product in 3% agarose gel to detect the result, and recovering the insert band by using a DNA gel recovery and purification kit. A59 bp fragment was obtained, consistent with the expected size.

The PCR-recovered product was ligated with the plasmid vector pcDNA3.1(+)

The PCR recovered fragment and the plasmid vector pcDNA3.1(+) are cut by NheI and HindIII enzyme and then are connected by T4DNA ligase, the connection product is transformed into a chemically competent cell DH5a strain and is coated on a plate of an ampicillin resistant solid culture medium, a plurality of monoclonal colonies are picked and inoculated into an ampicillin resistant liquid culture medium, and the ampicillin resistant liquid culture medium is subjected to shaking culture at the constant temperature of 37 ℃ and 250rpm overnight. And extracting the plasmid by using a plasmid extraction kit to obtain a recombinant plasmid vector pNC 1.

Identification of recombinant plasmid vector pNC1

The NheI and HindIII enzyme digestion is used for identification, and the enzyme digestion identification system and the reaction conditions are as follows:

recombinant plasmid DNA 5. mu.L, 10 Xbuffer 2. mu.L, NheI 0.5. mu.L, HindIII 0.5. mu.L, ddH2O 11. mu.L

The total volume was 20. mu.L. 37 ℃ for 15 min.

And (5) enzyme digestion identification is carried out, and the obtained product is consistent with the expected analysis size.

And (3) selecting positive clones which are subjected to enzyme digestion verification to carry out sequencing analysis, and verifying the correctness of the recombinant plasmid vector by FinchTV analysis and BLAST software comparison. The analysis result shows that the nucleotide sequence of the enhancer is completely correct, and the recombinant plasmid vector pNC1 is successfully obtained.

FGF2-pcDNA3.1(+) construct, FGF2-pNC1 construct and his-DnaE-FGF2-pNC1 construct

The DNA sequence (amino acids 143-288, PRO-0000008933) of the fgf2 gene (i.e., the mature functional fragment of the fgf2 gene) without the propeptide sequence was designed for codon optimization in humans.

The designed FGF2 gene was synthesized using GeneArt Strings Gene Synthesis service (Thermo-Fisher Scientific, Waltham, Mass.), and the synthesized FGF2 gene was cloned into pcDNA3.1(+) and pNC1 with EcoRI and NotI sites to form the FGF2-pcDNA3.1(+) construct and the FGF2-pNC1 construct, as shown in FIGS. 1A,1B 2. All sequences were confirmed by Sanger sequencing.

To optimize purification, the present invention uses the 6x his tag and uses the DNA polymerase iii (dnae) intein of Nostoc punctiforme (Nostoc punctiforme) PCC73102(Npu) fused with the FGF2 gene to facilitate purification of human fibroblast growth factor 2(FGF 2).

Designed fgf2 and 6x his-Npu DnaE intein genes were synthesized using GeneArt strands gene synthesis service (Thermo-Fisher Scientific, Waltham, MA). The 6x his-Npu DnaE intein was fused to fgf2 by overlap PCR.

To further increase expression of fgf2, an enhancer sequence containing NF-. kappa.B binding sites and CREB binding sites was synthesized using the GeneArt Strings Gene Synthesis service and cloned into pcDNA3.1(+) with NheI and HindIII sites (Thermo-Fisher Scientific, Waltham, Mass)) to form the expression plasmid vector pNC 1. Synthetic 6xhis-DnaE-FGF2) was cloned into pNC1 with EcoRI and NotI sites to form the his-DnaE-FGF2-pNC1 construct, as shown in fig. 1C. All sequences were confirmed by Sanger sequencing. Antibodies used for western blotting: mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), mouse anti-beta-actin (Sigma, St. Louis, MO).

The specific process of constructing the FGF2-pcDNA3.1(+) construct is as follows:

fgf2 Gene PCR amplification

fgf2 the template was synthesized by GeneArt Stringservice (thermos scientific), and its DNA sequence is shown in SEQ ID NO.1

CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCA CCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATG GTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGC GTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAG CTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAA TAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTA AGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCA AACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGT ACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAA AACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTG CCAAGTCTTAATAATAA are provided. The forward and reverse primers used are shown in Table 1. The following reactants were added to the EP tube in order: mu.l of template, 1 Xbuffer, 1. mu.M forward primer, 1. mu.M reverse primer, 0.5. mu.l polymerase, 4. mu.l dNTP, and ddH2O to 50. mu.l of reaction system.

Reaction conditions are as follows: 95 deg.C for 5 min; 95 ℃ for 15 s; 55 ℃ for 10 s; 72 ℃ for 30 min; 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃.

And (3) carrying out electrophoresis on the PCR amplification product in 1% agarose gel to detect the result, and recovering the insert band by using a DNA gel recovery and purification kit. A460 bp fragment was obtained, consistent with the expected size.

The PCR-recovered product was ligated with the plasmid vector pcDNA3.1(+)

The PCR recovered fragment and the plasmid vector pcDNA3.1(+) are cut by EcoRI and NotI and then are connected by T4DNA ligase, the connection product is transformed into a chemically competent cell DH5a strain and is coated on a plate of an ampicillin resistant solid culture medium, a plurality of monoclonal colonies are picked and inoculated into an ampicillin resistant liquid culture medium, and the mixture is subjected to shaking culture at 37 ℃ and 250rpm overnight by a constant temperature shaking table. Extracting the plasmid by using a plasmid extraction kit to obtain the FGF2-pcDNA3.1(+) construct.

Identification of FGF2-pcDNA3.1(+) construct

The restriction enzyme digestion is carried out by EcoRI and NotI, and the restriction enzyme digestion identification system and the reaction conditions are as follows:

FGF2-pcDNA3.1(+) construct 5. mu.L, 10 Xbuffer 2. mu.L, EcoRI 0.5. mu.L, NotI 0.5. mu.L, ddH2O 11. mu.L in total volume 20. mu.L. 37 ℃ for 15 min.

And selecting a positive clone with correct enzyme digestion verification for sequencing analysis, and comparing the result by FinchTVV analysis and BLAST software to verify the correctness of the FGF2-pcDNA3.1(+) construct. The analysis result shows that the FGF2 nucleotide sequence is completely correct, and the FGF2-pcDNA3.1(+) construct is successfully obtained.

The construction of the FGF2-pNC1 construct is specifically as follows:

fgf2 Gene PCR amplification

fgf2, the DNA sequence of which is shown in SEQ ID NO. 1:

CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAG GACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGG ATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTAC GGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGA AGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGG TACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTG CGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACA ATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGT TGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACA GGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAA GTCTTAATAATAA are provided. The forward and reverse primers used are shown in Table 1. The following reactants were added to the EP tube in order: mu.l of template, 1 Xbuffer, 1. mu.M forward primer, 1. mu.M reverse primer, 0.5. mu.l polymerase, 4. mu.l dNTP, and ddH2O to 50. mu.l of reaction system.

Reaction conditions are as follows: 95 deg.C for 5 min; 95 ℃ for 15 s; 55 ℃ for 10 s; 72 ℃ for 30 min; 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃. And (3) carrying out electrophoresis on the PCR amplification product in 1% agarose gel to detect the result, and recovering the insert band by using a DNA gel recovery and purification kit. A460 bp fragment was obtained, consistent with the expected size.

The PCR-recovered product was ligated with the plasmid vector pNC1

The PCR recovered fragment and the plasmid vector pNC1 were digested with EcoRI and NotI, ligated with T4DNA ligase, the ligation product was transformed into chemically competent cell strain DH5a, spread on a plate of ampicillin resistant solid medium, several monoclonal colonies were picked up and inoculated into ampicillin resistant liquid medium, and shake-cultured overnight at 37 ℃ and 250rpm on a constant temperature shaker. Plasmids were extracted using a plasmid extraction kit to obtain pNC1 constructs.

Identification of FGF2-pNC1 construct

FGF2-pNC1 construct 5. mu.L, 10 Xbuffer 2. mu.L, EcoRI 0.5. mu.L, NotI 0.5. mu.L, ddH₂O11. mu.L in a total volume of 20. mu.L. 37 ℃ for 15 min. And (5) enzyme digestion identification is carried out, and the obtained product is consistent with the expected analysis size.

And selecting positive clones with correct enzyme digestion verification for sequencing analysis, and comparing the results through FinchTV analysis and BLAST software to verify the correctness of the FGF2-pNC1 construct. The analysis result shows that the FGF2 nucleotide sequence is completely correct, and the FGF2-pNC1 construct is successfully obtained.

The specific process for constructing the his-DnaE-FGF2-pNC1 construct is as follows:

fusion PCR amplification of his-DnaE-FGF2

The template of his-DnaE-FGF2 was synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO. 2:

CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCC TGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGA GAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGC AACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCG AGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGAT CAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAG ATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGA TGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGA GATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATC GTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACA ACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAG GGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGC CTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACG GCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGA CCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAG GATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATC CGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATC CATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATC CGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGT TAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAA GAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGT GCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTAT AGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGA CCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAA GGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA are provided. The forward and reverse primers used with the His-DnaE forward primer and FGF2 reverse primer are shown in Table 1. The following reactants were added to the EP tube in order: mu.l of template, 1 Xbuffer, 1. mu.M forward primer, 1. mu.M reverse primer, 0.5. mu.l polymerase, 4. mu.l dNTP, and ddH2O to 50. mu.l of reaction system.

Reaction conditions are as follows: 95 deg.C for 5 min; 95 ℃ for 15 s; 55 ℃ for 10 s; 72 ℃ for 30 min; 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃. And (3) carrying out electrophoresis on the PCR amplification product in 1% agarose gel to detect the result, and recovering a GFP strip by using a DNA gel recovery and purification kit. A1097 bp fragment was obtained, consistent with the expected size.

The PCR-recovered product was ligated with the plasmid vector his-DnaE-FGF2

The PCR recovered fragment and the plasmid vector his-DnaE-FGF2 are cut by EcoRI and NotI, then are connected by T4DNA ligase, the connection product is transformed into a chemically competent cell DH5a strain, the strain is coated on a plate of an ampicillin resistant solid culture medium, a plurality of monoclonal colonies are picked up and inoculated into an ampicillin resistant liquid culture medium, and the ampicillin resistant liquid culture medium is subjected to shaking culture at the constant temperature of 37 ℃ and 250rpm overnight. And extracting the plasmid by using a plasmid extraction kit to obtain the his-DnaE-FGF2 construction body.

Identification of his-DnaE-FGF2 construct

the his-DnaE-FGF2 construct 5. mu.L, 10 Xbuffer 2. mu.L, EcoRI 0.5. mu.L, NotI 0.5. mu.L, ddH2O 11. mu.L in total volume 20. mu.L. 37 ℃ for 15 min.

And selecting positive clones with correct enzyme digestion verification for sequencing analysis, and comparing the results with FinchTV analysis and BLAST software to verify the correctness of the his-DnaE-FGF2 construct. The analysis result shows that the his-DnaE-FGF2 nucleotide sequence is completely correct, and the his-DnaE-FGF2 construct is successfully obtained.

TABLE 1

The DNA containing NheI and HindIII restriction site enhancers synthesized by PCR was SEQ ID NO.11 (i.e., SEQ ID NO.5 containing NheI and HindIII restriction sites):

GCTAGCGGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAAC ACTGCTCAACAAGCTT。

the DNA sequence of FGF2 synthesized by PCR and containing restriction sites for EcoRI and NotI is SEQ ID NO.12 (i.e., SEQ ID NO.1 containing restriction sites for EcoRI and NotI):

GAATTCCCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCC ACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAAT GGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGG CGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAA GCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTA ATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCT AAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTC AAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGG TACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCA AAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGT GCCAAGTCTTAATAATAAGCGGCCGC。

the DNA sequence of the PCR-synthesized his-DnaE-FGF2 with restriction sites for EcoRI and NotI is SEQ ID NO.13 (i.e., SEQ ID NO.2 with restriction sites for EcoRI and NotI):

GAATTCACCATGCATCATCACCATCACCACGCCGAGTACTACGA GACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGC AAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGG ACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCA CGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGAC GGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCG TGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGA GCTGGACCTGATGAGGGTGGACAACCTGCCCAACGCCGAGTAC TACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCA TCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAG CGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAG TGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGG AGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCAT GACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAG AGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACCCGG CTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACA CTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTT TCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGA GAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAA CGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACC TTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGT GACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATT ATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGC CCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGC CCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTC TTAATAATAAGCGGCCGC。

cell culture and transfection

HEK293T and C2C12 cells were maintained in DMEM containing 10 vol% FBS and 1 vol% penicillin streptomycin solution (Thermo-Fisher Scientific, Waltham, MA) at ambient temperature of 37 ℃ with 5% CO 2. PC12 cells were maintained in DMEM containing 10 vol% HS, 5 vol% FBS and 1 vol% penicillin streptomycin solution at ambient temperature of 37 ℃ containing 5% CO 2. pcDNA3.1-FGF2, pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transfected into HEK293T using Lipofectamine 2000(Thermo-Fisher Scientific, Waltham, Mass.) according to the manufacturer's instructions.

Purification of FGF2

The medium was collected and spun at 2,000g for 10 minutes to remove cell debris and filtered through a 0.45 μm filter. The filtrate was then passed through a pre-packed heparin-agarose column (BioRad Laboratories, Hercules, Calif.) equilibrated with 50mM Tris-HCl (pH 7.5). The column was washed thoroughly with 50mM Tris-HCl (pH7.5), 0.2M NaCl. FGF2 was eluted with a NaCl gradient from 0.3M to 3M (4-5 bed volumes for gradient). After elution, the protein was passed through a pre-packed Sephadex G25 column equilibrated with 50mM Tris-HCl (pH7.5) and eluted with the same buffer.

For intracellular expressed FGF2, cells were washed three times with ice-cold TBS and then sonicated in lysis buffer (TBS supplied with a Cocktail of clomplete tm Protease inhibitors (clomplete tm Protease Inhibitor Cocktail)). FGF2 was then purified in culture medium.

To obtain intein cleaved FGF2, histidine-tagged DnaE-FGF2 was purified by prepackaged Ni-NTA column. After washing, the column was incubated in 50mM Tris-HCl (pH6.2), 10mM EDTA, 200mM NaCl at 22 ℃ for different durations of 0, 1, 2, 4, 6, 10 hours, respectively, to induce C-terminal cleavage of the DnaE intein.

Protein analysis

Proteins were separated on a 15% by volume Tris-glycine SDS-PAGE. The gel was stained with silver to obtain purified protein. On the gel, the band corresponding to FGF2 was excised, washed and incubated with 1. mu.g trypsin in 50mM NH 4HCO 3 overnight at 4 ℃. The hydrolyzed samples were analyzed by LTQ Velos Linear Ion Trap Mass Spectrometers (Thermo Fisher scientific, San Jose) coupled to an Accela HPLE system. Complete MS scans (300-. For cell lysates, samples were spotted or transferred onto 0.2 μmNC membranes (BioRad Laboratories, Hercules, CA) and then blotted with antibodies (mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), mouse anti- β -actin (Sigma, st. louis, MO)).

Biological assay for FGF2

To monitor the effect of FGF2 on cell proliferation, MTT assays were performed as previously described. Briefly, C2C12 cells were seeded in 96-well plates and DMEM +0.5 vol% FBS and 1ng/mL of commercially available FGF2, purified FGF2 and endopeptidase cleaved FGF2 were provided. The activity of the cells was determined by adding MTT to a final concentration of 1mg/mL and incubated at 37 ℃ for 6 hours. The medium was then replaced with DMSO and the absorbance measured at 540nm in a microplate reader.

To test the neurotrophic effect of FGF2, PC12 cells were cultured for 3 days with medium supplied with 2ng/mL of FGF 2. The morphology of the cells was observed, and images were taken by phase contrast optical microscopy.

Results

Construction of a plasmid expressing human identical exogenous FGF2

The full length FGF2 protein consists of 288 amino acids, with amino acids 1-142 cleaved and removed to yield functional FGF 2. Thus, the protein sequence of amino acid 143-288 was codon optimized for Homo sapiens (Homo sapiens). To explore the feasibility of maximizing protein expression, the NF-. kappa.B binding site and CREB binding site enhancer sequences were cloned into pcDNA3.1 vector containing the native CMV immediate early promoter/enhancer sequence. The synthesized fgf2 gene was then cloned into pcDNA3.1 and pNC1 vectors (FIG. 1) for comparison of expression of the two clones in mammalian cells. Experiments have shown that the pNC1 vector greatly increased the expression level of FGF2 in mammalian cells, as shown in fig. 2. The neomycin resistance gene also provides a selectable marker by G418 sulfate. Stably transfected cells can thus be selected for purification of human identical exogenous FGF 2.

Expression of soluble FGF2 in HEK293T cells

HEK293T cells were selected for purification because of their ease of transfection. The SV40 large T antigen also provides the ability of cells to replicate transfection plasmids, which contain the SV40 origin of replication. Furthermore, the CMV promoter is one of the strongest promoters and is constitutively active in HEK293T cells. While HEK293T cells are adherent cells, their suitability for suspension culture is versatile and therefore advantageous for expanding the production of FGF 2. The pcDNA3.1-FGF2pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transiently transfected into HEK293T cells and expression was monitored. Experiments show that: the pNC1 vector greatly increased the expression level of FGF2 (fig. 2A, 2B). Expression of FGF2 peaked 48 hours after transfection and remained stable at longer time points.

One of the drawbacks of protein preparation and purification using bacterial systems is the formation of insoluble aggregates. To test the solubility of FGF2 expressed in HEK293T cells, pellet fractions were harvested from cell lysates. The insoluble fraction was sonicated in Laemmili sample buffer and then electrophoresed in SDS-PAGE. Western blot of lysates showed that most of FGF2 was soluble (fig. 2C). Compared with the FGF2 standard, FGF2 expressed in pcDNA3.1-FGF2 was about 20 μ g/mL, and FGF2 expressed in pNC1-FGF2 was about 50 μ g/mL.

Purification and protein sequencing of FGF2

To maximize the yield of purified FGF2, HEK293T cells were transfected with pNC1-FGF 2. 48 hours after transfection, cells were harvested and lysed to purify FGF2 expressed intracellularly. FGF2 was purified by heparin-agarose affinity chromatography followed by size exclusion chromatography. Eluted FGF2 was electrophoresed on 15% SDS-PAGE (FIG. 3A). Samples were lyophilized and reconstituted in 0.1x PBS. To check the purity of FGF2, silver staining was performed after electrophoresis and showed very high purity, only 1 weak band was found above purified FGF2 (fig. 3B). The yield was also satisfactory. To identify purified FGF2, bands on SDS-PAGE were excised, solubilized, and analyzed by LC-MS after trypsinization. Sequencing results showed that the primary structure of purified FGF2 was identical to mature exogenous human FGF2 (table 2). Thus, we expect purified FGF2 to exhibit the same properties, activities, and functions as the human natural counterpart.

FGF2 expressed by HEK293T has biological activity

FGF2 has been shown to stimulate cell growth in a number of cell types. After confirming the primary structure of purified FGF2, we treated cultured C2C12 with 1ng/mL of purified and commercially available FGF2, PBS serving as a control. The viability of the cultured cells was then measured by MTT assay (fig. 4A). Both FGF2 samples were able to stimulate proliferation of C2C12 cells. Purified FGF2 showed similar or even higher biological activity than commercial FGF2, but the difference was not significant. FGF2 also exhibits neurotrophic activity in neuronal cell lines. Thus, the neurotrophic activity of purified FGF2 was tested in PC12 cells. After 3 days of FGF2 treatment, prolonged neurite outgrowth was observed, which was absent in the PBS control group (fig. 4B). The data demonstrate that purified FGF2 is biologically active and has activity similar to that of commercially available FGF 2.

Intein-mediated purification in HEK293T cells

A recombinant protein cascade containing 6 × histidine-tagged DnaE-FGF2 was cloned into pNC1 vector (FIG. 1C) and then transfected into HEK293T cells. The medium was collected and spun at 2,000g for 10 minutes to remove cell debris and filtered through a 0.45 μm filter. The protein was purified by Ni-NTA affinity chromatography. FGF2 excision was performed on Ni-NTA columns with lysis buffer (50mM Tris-HCl pH6.2, 10mM EDTA, 200mM NaCl) at different time points at 22 ℃. The beads and supernatant were boiled in 2x Laemmili sample buffer to elute bead-bound proteins, followed by SDS-PAGE (fig. 5). The experimental results show that complete cleavage of inteins requires at least 5 hours of incubation. The identity of the cleaved protein was determined by LC-MS and the primary sequence of the cleaved FGF2 was found to be identical to mature human FGF2 (table 3). The biological activity of cleaved FGF2 on C2C12 cells was also tested, and the performance of endoproteolytic cleaved FGF2 was also identical to purified FGF2 (fig. 6A, 6B).

TABLE 2

Liquid chromatography-tandem mass spectrometry analysis of purified FGF2

^aAfter trypsin partial digestion of purified FGF2, the N-terminal and C-terminal sequences were identified by Mascot search engine

^bTheoretical mass to charge ratio of peptide

^cExperimental Mass to Charge ratios of peptides

TABLE 3

Analysis of intein-cleaved FGF2 by liquid chromatography-tandem mass spectrometry

^bTheoretical mass to charge ratio of peptide

^cExperimental Mass to Charge ratios of peptides

FGF2 is a highly valuable protein with a wide range of potency including angiogenesis, neurogenesis and wound healing. One of the reasons impeding the research of the medical use of FGF2 is the high cost of purification and bioactive FGF 2. Using HEK293T cells, we successfully expressed and purified human FGF2 (fig. 3). The silver stained gel proved to be very satisfactory in purity. Purified FGF2 showed the same mitogenic or neurotrophic activity as commercially available FGF2 in C2C12 and PC12 cell cultures. This simple protocol allows for laboratory scale production of human exogenous FGF2 in mammalian systems and can be easily scaled up for large scale production.

One of the most widely used host systems for FGF2 purification is bacteria. However, simple prokaryotes lack the necessary post-translational modifications, including splicing, glycosylation and disulfide bonding, activity and solubility of the protein for purification. The natural folding of the purified protein is critical to its function and solubility. Protein aggregates or inclusion bodies are often found when eukaryotic proteins are expressed in bacterial systems. Methods involving inducing proteins or denaturing and renaturing protein aggregates at lower temperatures do not always produce good yields. Another problem with bacterial systems is endotoxins. Lipopolysaccharides or endotoxins are usually present in E.coli. The presence of endotoxin triggers an immune response in humans and may lead to septic shock. Although there are commercially available kits and protocols for removing endotoxin, contamination with endotoxin is generally unavoidable. However, the above problems can be easily overcome using mammalian cells.

Using mammalian cells to express proteins, we were able to purify functional, correctly folded and modified proteins (table 2, fig. 3, fig. 4). Therefore, we succeeded in purifying unlabeled human mature exogenous FGF2 in mammalian cells for the first time.

In the present invention, we have demonstrated that DnaE is a fast-cutting intein in HEK293T cells. By using this intein we have demonstrated that the primary structure (table 3) and biological activity (figure 6) of the cleaved FGF2 is identical to its natural counterpart. The medical use of purified FGF2 will be extensive. In addition, the plasmid vector pNC1 can be used to express a variety of valuable proteins.

Sequence listing

<110> root of Bell Tree

<120> production of human embryonic cytokine-1 by using optimized FGF2 gene functional region sequence

<130> 190064882

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 447

<212> DNA

<213> Artificial Synthesis

<400> 1

ccggctctcc cagaggatgg cggctcagga gcctttccac caggacactt caaagatccg 60

aagaggcttt actgcaagaa tggtggattt ttcctccgca tccatccaga cggtcgggtg 120

gacggcgtac gggagaaatc cgatccgcat ataaagctgc agctgcaagc tgaagaacga 180

ggggtggtta gcataaaggg cgtgtgtgct aataggtacc ttgccatgaa agaagacgga 240

cggctcctcg cttctaagtg cgtgaccgac gagtgcttct tctttgagcg gctagagtca 300

aacaattata acacctatag gtcaagaaag tatacgagct ggtacgttgc ccttaagcgg 360

accggccagt acaagcttgg tagcaaaaca ggccctggcc agaaggctat cctcttcctc 420

cctatgagtgccaagtcttaataataa 447

<210> 2

<211> 1077

<212> DNA

<213> Artificial Synthesis

<400> 2

catcatcacc atcaccacgc cgagtactac gagaccgaga tcctgaccgt ggagtacggc 60

ctgctgccca tcggcaagat cgtggagaag aggatcgagt gcaccgtgta cagcgtggac 120

aacaacggca acatctacac ccagcccgtg gcccagtggc acgacagggg cgagcaggag 180

gtgttcgagt actgcctgga ggacggcagc ctgatcaggg ccaccaagga ccacaagttc 240

atgaccgtgg acggccagat gctgcccatc gacgagatct tcgagaggga gctggacctg 300

atgagggtgg acaacctgcc caacgccgag tactacgaga ccgagatcct gaccgtggag 360

tacggcctgc tgcccatcgg caagatcgtg gagaagagga tcgagtgcac cgtgtacagc 420

gtggacaaca acggcaacat ctacacccag cccgtggccc agtggcacga caggggcgag 480

caggaggtgt tcgagtactg cctggaggac ggcagcctga tcagggccac caaggaccac 540

aagttcatga ccgtggacgg ccagatgctg cccatcgacg agatcttcga gagggagctg 600

gacctgatga gggtggacaa cctgcccaac ccggctctcc cagaggatgg cggctcagga 660

gcctttccac caggacactt caaagatccg aagaggcttt actgcaagaa tggtggattt 720

ttcctccgca tccatccaga cggtcgggtg gacggcgtac gggagaaatc cgatccgcat 780

ataaagctgc agctgcaagc tgaagaacga ggggtggtta gcataaaggg cgtgtgtgct 840

aataggtacc ttgccatgaa agaagacgga cggctcctcg cttctaagtg cgtgaccgac 900

gagtgcttct tctttgagcg gctagagtca aacaattata acacctatag gtcaagaaag 960

tatacgagct ggtacgttgc ccttaagcgg accggccagt acaagcttgg tagcaaaaca 1020

ggccctggcc agaaggctat cctcttcctc cctatgagtg ccaagtctta ataataa 1077

<210> 3

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 3

ggaaatcccc ggaaatcccc 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 4

tgcgtcaaca ctgctcaac 19

<210> 5

<211> 47

<212> DNA

<213> Artificial Synthesis

<400> 5

ggaaatcccc ggaaatcccc gtaaaatttg cgtcaacact gctcaac 47

<210> 6

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 6

aaaagctagc ggaaatcccc ggaaatcccc 30

<210> 7

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 7

aaaaaagctt gttgagcagt gttgacgca 29

<210> 8

<211> 32

<212> DNA

<213> Artificial Synthesis

<400> 8

aaagaattcc caccatgcat catcaccacc at 32

<210> 9

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 9

aaagcggccg cttattatta agacttggca c 31

<210> 10

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 10

aaaagaattc gccaccatgc cggctctccc a 31

<210> 11

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 11

gctagcggaa atccccggaa atccccgtaa aatttgcgtc aacactgctc aacaagctt 59

<210> 12

<211> 461

<212> DNA

<213> Artificial Synthesis

<400> 12

gaattcccgg ctctcccaga ggatggcggc tcaggagcct ttccaccagg acacttcaaa 60

gatccgaaga ggctttactg caagaatggt ggatttttcc tccgcatcca tccagacggt 120

cgggtggacg gcgtacggga gaaatccgat ccgcatataa agctgcagct gcaagctgaa 180

gaacgagggg tggttagcat aaagggcgtg tgtgctaata ggtaccttgc catgaaagaa 240

gacggacggc tcctcgcttc taagtgcgtg accgacgagt gcttcttctt tgagcggcta 300

gagtcaaaca attataacac ctataggtca agaaagtata cgagctggta cgttgccctt 360

aagcggaccg gccagtacaa gcttggtagc aaaacaggcc ctggccagaa ggctatcctc 420

ttcctcccta tgagtgccaa gtcttaataa taagcggccg c 461

<210> 13

<211> 1097

<212> DNA

<213> Artificial Synthesis

<400> 13

gaattcacca tgcatcatca ccatcaccac gccgagtact acgagaccga gatcctgacc 60

gtggagtacg gcctgctgcc catcggcaag atcgtggaga agaggatcga gtgcaccgtg 120

tacagcgtgg acaacaacgg caacatctac acccagcccg tggcccagtg gcacgacagg 180

ggcgagcagg aggtgttcga gtactgcctg gaggacggca gcctgatcag ggccaccaag 240

gaccacaagt tcatgaccgt ggacggccag atgctgccca tcgacgagat cttcgagagg 300

gagctggacc tgatgagggt ggacaacctg cccaacgccg agtactacga gaccgagatc 360

ctgaccgtgg agtacggcct gctgcccatc ggcaagatcg tggagaagag gatcgagtgc 420

accgtgtaca gcgtggacaa caacggcaac atctacaccc agcccgtggc ccagtggcac 480

gacaggggcg agcaggaggt gttcgagtac tgcctggagg acggcagcct gatcagggcc 540

accaaggacc acaagttcat gaccgtggac ggccagatgc tgcccatcga cgagatcttc 600

gagagggagc tggacctgat gagggtggac aacctgccca acccggctct cccagaggat 660

ggcggctcag gagcctttcc accaggacac ttcaaagatc cgaagaggct ttactgcaag 720

aatggtggat ttttcctccg catccatcca gacggtcggg tggacggcgt acgggagaaa 780

tccgatccgc atataaagct gcagctgcaa gctgaagaac gaggggtggt tagcataaag 840

ggcgtgtgtg ctaataggta ccttgccatg aaagaagacg gacggctcct cgcttctaag 900

tgcgtgaccg acgagtgctt cttctttgag cggctagagt caaacaatta taacacctat 960

aggtcaagaa agtatacgag ctggtacgtt gcccttaagc ggaccggcca gtacaagctt 1020

ggtagcaaaa caggccctgg ccagaaggct atcctcttcc tccctatgag tgccaagtct 1080

taataataagcggccgc 1097

Claims

1. A construct comprising the DNA sequence shown in SEQ ID No.2 between the EcoRI and NotI restriction sites of a plasmid vector pNC1, said pNC1 being formed by cloning the NF- κ B binding site and the CREB binding site behind the CMV promoter of pcDNA3.1 (+).

2. A method of making a construct, comprising the steps of:

a. synthesizing a DNA sequence shown in SEQ ID NO.2 containing EcoRI and NotI restriction enzyme cutting sites;

c. connecting the DNA sequence obtained in the step a with the large fragment obtained in the step b by adopting ligase;

d. c, transfecting the cell with the ligation product obtained in the step c, screening positive clones, and extracting plasmids;

wherein the plasmid vector is pcDNA3.1(+) or pNC1, the pNC1 is formed by cloning the NF-. kappa.B binding site and the CREB binding site behind the CMV promoter of pcDNA3.1 (+).

3. Use of the construct of claim 1 for expressing FGF-2.

4. A method of preparing FGF-2, the method comprising:

a. culturing the cells: culturing a mammalian cell;

b. transfection: transfecting the mammalian cell obtained in step a with the construct of claim 1;

c. and (3) purification: FGF-2 was purified by affinity chromatography.

5. The method for the preparation of FGF-2 according to claim 4, wherein the affinity chromatography is performed by a heparin-binding domain.

6. The method for preparing FGF-2 according to claim 4, wherein in step b, the mammalian cells obtained in step a are transfected with the construct of claim 1; the affinity chromatography is mediated by the intein DnaE.

7. The method for producing FGF-2 according to any of claims 4 to 6, wherein the mammalian cell is HEK 293T.