A fusion FGF1 protein sequence and a corresponding DNA sequence which do not affect the ability to bind to heparin

Abstract

The application discloses a fusion FGF1 protein sequence which does not affect the heparin binding capacity and a corresponding DNA sequence, and belongs to the technical field of biology.The nucleotide sequence of the fusion protein FGF1-Pt provided by the application is shown in Seq ID No.10, and the amino acid sequence is shown in Seq ID No.5.The application places Pt at the C-terminal of FGF1, has the best heparin binding capacity without adding a linking peptide, has the highest expression amount of Escherichia coli, and provides materials for the function research of the membrane penetrating peptide of Pt and how Pt affects the spatial structure and activity of FGF1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a fusion FGF1 protein sequence and its corresponding DNA sequence that do not affect the ability to bind to heparin. Background Technology

[0002] Biological macromolecular drugs are widely used to treat major diseases such as diabetes, cancer, and cardiovascular diseases. Due to their high specificity, biological macromolecular drugs have significant advantages in treating these major diseases and are globally recognized as one of the most promising high-end fields in 21st-century drug development. Despite the increasing importance of biological macromolecular drugs, many challenges and obstacles remain in their application, such as the difficulty in effectively crossing biological barriers within the body. Therefore, improving the delivery of biological macromolecular drugs to target sites and cells is a crucial issue that urgently needs to be addressed to achieve efficient delivery of these drugs.

[0003] Cell-penetrating peptides (CPPs) are a class of short peptides capable of penetrating cell membranes. They typically consist of no more than 30 amino acid residues, all carrying a net positive charge. They can interact with the cell membrane to penetrate it and can carry polypeptides, proteins, and nucleic acid molecules into the cell. Cell-penetrating peptides are functional molecules that have been extensively studied in recent years, especially in the fields of pharmacology and therapeutics, providing new research ideas for drug delivery and targeted therapy (Chen Y et al. 2006. Transdermal protein delivery by a co-administered peptide identified via phage display. Nature Biotechnology. 24:455-460); Guidotti G et al. 2017. Cell-Penetrating Peptides: From Basic Research to Clinics. Trends Pharmacol Sci. 38:406-424; Zhou M et al. 2022. The role of cell-penetrating peptides in potential anti-cancer therapy. Clin Transl Med. 12:e822; Zorko M, Langel 2022. Cell-Penetrating Peptides. Methods Mol Biol. 2383:3-32; Chen Songbin et al. 2016. Application of a fusion protein in cosmetics, Chinese Patent No. CN201610026298; Ma Jie et al. 2017. Preparation of TAT-hEGF fusion protein and its application in invisible masks, Chinese Patent No. CN102017001470311).

[0004] Drosophila antennal protein ANTP contains 378 amino acid residues (P02833). The 16 amino acid sequences from residues 339 to 354 (RQIKIWFQNRRMKWKK) have been shown to have the function of penetrating the cell membrane and are called Penetratin or Pt. Penetrants (Pt) are a highly representative type of cell-penetrating peptides, capable of carrying bioactive molecules such as proteins and nucleic acids into the cell membrane, and have promising applications in cell biology and pharmacology (Derossi D et al. 1994. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 269:10444-10450; Dupont E et al. 2015. Penetrant Story: An overview. Methods Mol Biol. (Review) 1324:29-37; Gehan P et al. 2020. Penetrant translocation mechanism through asymmetric droplet interface bilayers. Biochim Biophys Acta Biomembr. 1862:183415).

[0005] Acidic fibroblast growth factor (FGF1) is a growth factor polypeptide that can bind to heparin. The mature peptide in the human body contains 140 amino acid residues, with a heparin-binding amino acid sequence near the carboxyl terminus. Heparin binding is essential for FGF1 to function. FGF1 is expressed at higher levels in the kidneys and brain, and at lower levels in the heart and skeletal muscle. Its function is believed to involve embryonic development, wound healing, neuronal regeneration, and angiogenesis. In recent years, FGF1 has also been shown to be useful in treating hyperglycemia in diabetes. Studies have shown that FGF1 exerts its hypoglycemic effect by inhibiting the hypothalamic-pituitary-adrenal axis, reducing hepatic acetyl-CoA synthesis, enhancing insulin sensitivity, and through central mechanisms. It may also promote insulin secretion and directly affect insulin levels (Suh JM et al. 2014. Endocrinization of FGF1 produces aneomorphic and potent insulin sensitizer. Nature. 513:436-9; Gasser E et al. 2017. FGF1-a new weapon to control type 2 diabetes mellitus. Nat Rev Endocrinol. 13:599-609; Leake I. 2019. Exploring the antidiabetic effects of FGF1. Nat Rev Endocrinol. 15:66; Sancar G et al. 2022. FGF1 and insulin control lipolysis by convergent pathways. Cell Metab. 34:171-183).

[0006] To develop and utilize FGF1, it is necessary to fuse the transmembrane peptide Pt with FGF1 and make corresponding changes to the linkage mode between Pt and FGF1 to evaluate the differences in the binding capacity of FGF1 and heparin caused by these changes. However, to date, no literature on Pt-FGF1 fusion proteins has been found. Summary of the Invention

[0007] The inventors fused Pt into FGF1 in various ways and conducted in-depth research on it. The fusion methods included: 1. placing Pt at the N-terminus or C-terminus of FGF1; 2. using 1) a non-linking peptide, 2) adding a rigid linking peptide, 3) or a flexible linking peptide between Pt and FGF1. Experiments demonstrated that the fusion protein FGF1-Pt (non-linking peptide, with Pt placed at the C-terminus) exhibits optimal heparin binding capacity and the highest expression level. Therefore, this invention provides a fused FGF1 protein sequence and its corresponding DNA sequence that do not affect heparin binding capacity. A review of domestic and international literature revealed no identical or similar sequences. The FGF1-Pt fusion protein of this invention is of great value for studying the retention of FGF1 function after Pt fusion and for researching Pt's membrane-penetrating function.

[0008] One of the objectives of this invention is to provide a fusion protein FGF1-Pt, the nucleotide sequence of which is shown in Seq ID No. 10.

[0009] Furthermore, the amino acid sequence of the fusion protein FGF1-Pt is shown in Seq ID No. 5.

[0010] The second objective of this invention is to provide a plasmid containing the Seq ID No. 10 sequence.

[0011] A third objective of this invention is to provide a host cell containing the aforementioned plasmid.

[0012] The fourth objective of this invention is to provide a method for preparing the fusion protein FGF1-Pt, wherein the method involves inserting the DNA encoding the fusion protein into an expression vector and introducing the vector into a prokaryotic expression system for expression.

[0013] The fifth objective of this invention is to provide the application of the above-mentioned fusion protein FGF1-Pt in the study of FGF1 or Pt function.

[0014] Furthermore, the application involves using the fusion protein FGF1-Pt to study cell membrane penetration and the influence of Pt on the spatial structure and activity of FGF1.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention places Pt at the C-terminus of FGF1. Without adding a linker peptide, it exhibits the best heparin binding ability and the highest expression level in E. coli. This provides materials for studying the transmembrane peptide function of Pt and how Pt affects the spatial structure and activity of FGF1. Attached Figure Description

[0017] Figure 1This is an electrophoresis image of the fusion protein CFE6 (FGF1-Pt) expressed by IPTG in Example 2.

[0018] Figure 2 This is an electrophoresis image of the fermented fusion protein CFE6 expressed in Example 3 after the first heparin column purification.

[0019] Figure 3 This is a gel electrophoresis image of the FGF1-Pt fusion protein CFE6 from Example 3 after a second heparin column purification.

[0020] Figure 4 This is an electrophoresis result of CFE6 concentrated using a heparin column in Example 4.

[0021] Figure 5 CFE2(Pt-FGF1) in Example 6 ΔNT24 Electrophoresis image after purification with heparin column. Detailed Implementation

[0022] Example 1: Construction of fusion protein expression plasmid and screening of expression strains

[0023] The FGF1 gene was constructed into the expression plasmid pET28, and the constructed plasmid was named pET28-BioA54.

[0024] The amino acid sequence of FGF1 is shown in Seq ID No. 1, and the DNA sequence encoding the above amino acids is shown in Seq ID No. 6 (lowercase text indicates plasmid sequence, underlined). catATG and ctcgag respectively The pET28 restriction sites are NdeI and XhoI.

[0025] The Drosophila antennal peptide ANTP contains 378 amino acid residues (P02833). A 16-amino acid sequence from amino acid R (339) to amino acid K (354) (RQIKIWFQNRRMKWKK) has been shown to have the function of penetrating the cell membrane and is called Penetratin or Pt. Its amino acid sequence is shown in Seq ID No. 2, and the DNA sequence encoding the above amino acids is shown in Seq ID No. 7.

[0026] To achieve fusion expression of FGF1 and Pt, a Pt codon-optimized DNA sequence (CFE6) was added to the carboxyl terminus based on the above sequence. This sequence includes Nde I and Xho I restriction sites at both ends, and the thickened sequence represents the DNA sequence encoding Pt. The fusion DNA sequence is shown in Seq ID No. 10:

[0027]

[0028] The amino acid sequence (CFE6) of the DNA-encoded fusion protein is as follows, where the first 141 amino acids are FGF1 and the 16 bolded amino acid residues are Pt.

[0029] Seq ID No. 5:

[0030]

[0031] To construct the FGF1-Pt fusion protein particle, primers were designed using pET28-BioA54 as a template, with the N-terminus digested with Nde I and the C-terminus digested with Xho I.

[0032] The target gene was obtained by three consecutive PCRs using primers T7 / CFE61, T7 / CFE62, and T7 / CFE63. After PCR, the gene was recovered by gel extraction, double digested with Nde I and Xho I, ligated with pET28 (Nde I / Xho I), and transformed into E. coli DH5α to obtain the transformant pET28-CFE6.

[0033] T7(Seq ID No.18):TAATACGACTCACTATAGGG;

[0034] CFE6-1 (Seq ID No. 19):AATTTTAATCTGACGGTCGGAAGACACCGG;

[0035] CFE6-2 (Seq ID No. 20): CCATTTCATGCGACGGTTCTGAAACCAAATTTTAATCTGACG;

[0036]

[0037] The pET28-CFE6 plasmid was transformed into Escherichia coli expression strain BL21(DE3) to obtain a strain expressing the fusion protein, which was named BL21-CFE6, and the expressed fusion protein was named CFE6.

[0038] Example 2: Induction of expression of fusion protein CFE6 in shake flasks and fermenters

[0039] 100 μL of BL21-CFE6 bacterial culture from Example 1 was inoculated into 5 ml of LB medium containing kanamycin and cultured at 37°C and 220 RPM for 3.5 h. 1 ml of the culture was then transferred to 50 ml of LB medium containing kanamycin (total 100 ml, two bottles) and cultured overnight at 30°C and 200 RPM to obtain the seed culture.

[0040] Prepare the fermentation tank culture medium with the following components: 12 g / L peptone, 12 g / L yeast extract, 0.5 g / L KH₂PO₄, 0.75 g / L K₂HPO₄, 0.5 g / L MgSO₄, and 2 g / L (NH₄)₂SO₄, dissolved in water to a volume of 3 L. Prepare the feed medium with the following components: 35 g / L glucose (based on the volume of the upper tank), dissolved in water to a volume of 500 ml. Sterilize the upper tank culture medium along with the fermentation tank, and sterilize the feed medium, feed piping, etc., with high-temperature, high-pressure steam sterilization at 115°C for 30 minutes. The next morning, install the fermentation equipment, and after debugging, inoculate.

[0041] Seed liquid concentration OD 600 The pH was 4.46. The fermenter temperature was adjusted to 37℃ and the pH to approximately 6.96 for inoculation, using about 60ml of seed culture. The remaining seed culture was transferred to a shake flask, and 3ml of the seed culture was inoculated into 300ml of LB medium containing kanamycin. The flask was placed in a shaker at 37℃ and a speed of 200 RPM for simultaneous incubation and induction with fermentation. After inoculation, the temperature was maintained at a constant 37℃ and the stirring speed at 200 RPM. Dissolved oxygen was used as a reference indicator to adjust the stirring speed and aeration rate, maintaining dissolved oxygen between 20% and 40%. Simultaneously, feeding was started at a rate of 100ml / h. The culture was incubated until the bacterial concentration exceeded 10 (OD200). 600 The measured value was cooled to 25°C, and IPTG (final concentration 100uM) was added for induction.

[0042] At 4.5 hours of cultivation, the bacterial concentration reached 10.45, and at this point, the concentration was 10.83 before cooling induction. Samples (40 μL) were taken hourly before and after fermentation induction, along with a 40 μL sample of the control culture in a shake flask. All samples were incubated with 20 μL of 3X Loading Buffer, boiled in a water bath for 3 minutes, and then subjected to electrophoresis. The results are shown below. Figure 1 As shown, by Figure 1 It can be seen that CFE6 was highly expressed, and the expression level in the fermenter after 5 hours of induction was significantly higher than that in the shake flask. Among them, Lanes: 1: before IPTG induction; 2: 1 h of induction; 3: 2 h of induction; 4: 3 h of induction; 5: 4 h of induction; 6: 5 h of induction; 7: 6 h of induction; 8: shake flask control; 9: Marker.

[0043] Example 3: Isolation and purification of fusion protein CFE6

[0044] (1) First heparin column purification

[0045] Take 1000 ml (approximately 100 g of bacterial cells) of the bacterial culture induced for 5 h in Example 2, dissolve completely, and then sonicate on ice at 80% power for 3 seconds, followed by a 7-second interval, for 30 minutes, repeating the sonication twice. Centrifuge the sonicated bacterial culture at 12000 RPM for 10 minutes and collect the supernatant. Dilute the supernatant with phosphate buffer (20 mM PB, 1 mM EDTA, pH 7.0) to a concentration of 50 g / L for heparin column purification. The total supernatant volume is 2 L, which is purified in two batches, with 1 L (approximately 50 g of bacterial cells) loaded each time.

[0046] CFE6 purification was performed using a heparin column (Heparin Nupharose FF) manufactured by Hangzhou Newlong Biotechnology Co., Ltd., with 50ml of column packing material and gravity-fed column tubes. The purification steps are as follows:

[0047] (1) Buffer preparation

[0048] Buffer A: 20mM PB, 1mM EDTA, pH7.0;

[0049] Buffer B: 20mM PB, 1mM EDTA, 200mM NaCl, pH7.0;

[0050] Buffer C: 20mM PB, 1mM EDTA, 750mM NaCl, pH7.0;

[0051] Buffer D: 20mM PB, 1mM EDTA, 1.5M NaCl, pH7.0;

[0052] Buffer E: 0.1M NaOH.

[0053] (2) Purification steps

[0054] ① Rinse the balance column with Buffer A;

[0055] ② The sample loading volume is 1L, and the flow-through is collected;

[0056] ③ Use Buffer A to balance the gravity column;

[0057] ④ Use Buffer B to wash away impurities;

[0058] ⑤ Rinse the gravity column with Buffer C;

[0059] ⑥ Use Buffer D to elute impurities;

[0060] ⑦ Rinse with Buffer E;

[0061] ⑧ Rinse with water until neutral;

[0062] ⑨ The flow rate for all steps is 10 ml / min.

[0063] After A 280nm The Buffer D elution buffer contained 90.66 mg of protein. Electrophoresis was performed to detect this protein, and the results are as follows: Figure 2 As shown, the SDS-PAGE was 12%, with Lines 1 and 2 having a loading volume of 5 μl; Lines 3, 4, 5, 6, and 7 having a loading volume of 10 μl; Lines: 1: supernatant sample; 2: flow-through; 3: Buffer A (20 mM PB, 1 mM EDTA, pH 7.0) elution; 4: Buffer B (20 mM PB, 1 mM EDTA, 200 mM NaCl, pH 7.0) elution; 5: Buffer C (20 mM PB, 1 mM EDTA, 750 mM NaCl, pH 7.0) elution; 6: Buffer D (20 mM PB, 1 mM EDTA, 1.5 M NaCl, pH 7.0) elution; 7: Buffer E (0.1 M NaOH) elution; 8: Marker.

[0064] The target protein CFE6 can bind to the heparin column and can be eluted with Buffer D (20mM PB, 1mM EDTA, 1.5M NaCl, pH 7.0). The resulting CFE6 has a purity greater than 80% and also contains a relatively large amount of low molecular weight impurities (Lane 6). The high content of the target protein in the flow-through may be due to the addition of an excessive amount of the target protein, causing an overload state (Lane 2).

[0065] (2) Second heparin column purification

[0066] Because Buffer D (20mM PB, 1mM EDTA, 1.5M NaCl, pH 7.0) eluent contained other contaminating proteins besides the primary target protein, 300ml (40.47mg) of this eluent was dialyzed into a buffer containing 20mM PB, 1mM EDTA, 150mM NaCl, pH 8.0. A small amount of turbidity appeared during dialysis. The following day, the dialyzed protein was centrifuged, and the OD280 value was measured. Using the same heparin purification column, the same buffer, and the same elution steps, 33mg of high-purity CFE6 was obtained, with a yield of 81.5%. Electrophoresis results are shown below. Figure 3 The SDS-PAGE concentration was 12%, and the loading volume was 10 μl / lane. The results showed that after the second heparin column purification, the purity of CFE6 exceeded 95%, and the Buffer AE solution was the same as that of the first heparin column purification.

[0067] Lanes: 1: CFE6 sample after first heparin column purification; 2: Flow-through; 3: Buffer A elution; 4: Buffer B elution; 5: Buffer C elution; 6: Buffer D elution 1; 7: Buffer D elution 2; 8: Buffer elution; 9: Marker.

[0068] Example 4: Concentration of CFE6 using heparin columns

[0069] Because the heparin packing material has a low loading capacity, the above purification method results in a large eluent volume and a low protein concentration. Therefore, the CFE6 solution previously processed in Example 3 was concentrated using different methods. Our company previously tested that the binding rate of heparin columns to CFE6 could reach 6 mg protein / ml packing material; therefore, we decided to use 30 ml of packing material to enrich and concentrate the previous CFE6.

[0070] (1) Sample

[0071] The protein elution buffer from the second heparin column purification was 600 ml, approximately 126 mg. It was diluted to 2600 ml with a buffer of 20 mM PB, 1 mM EDTA, pH 7.0. At this point, the NaCl concentration in the protein solution was approximately 350 mM, and the OD... 280 The value is 0.083, the concentration is 0.05 mg / ml, and the total is approximately 130 mg.

[0072] (2) Heparin purification column

[0073] 30m heparin column packing (Heparin Nupharose FF 20191108), column tube is 5×25cm medium pressure column.

[0074] (3) Buffer

[0075] Buffer A: 20mM PB, 1mM EDTA, pH7.0;

[0076] Buffer B: 20mM PB, 1mM EDTA, 1.5M NaCl, pH7.0;

[0077] Buffer C: 0.1M NaOH.

[0078] (4) Concentration Steps

[0079] ① Rinse the balance column with Buffer A;

[0080] ② Load the sample, collect the flow through, collect separately, and collect once every 500ml;

[0081] ③ Use Buffer A to balance the gravity column;

[0082] ④ Rinse with Buffer B;

[0083] ⑤ Rinse with Buffer C;

[0084] ⑥ Rinse with water until neutral;

[0085] ⑦ The flow rate throughout the process is 15 ml / min.

[0086] Electrophoresis detection: For each step of the above process, take 40 μL of sample, add 20 μL of 3× Loading Buffer, incubate in boiling water for 3 min, and then perform SDS-PAGE detection. See [link to SDS-PAGE analysis]. Figure 4 The sample was processed using SDS-PAGE at 12% with a loading volume of 10 μl per lane. Lanes: 1: Sample purified from the second heparin column; 2: Flow-through 1; 3: Flow-through 2; 4: Flow-through 3; 5: Flow-through 4; 6: Flow-through 5; 7: Buffer B elution 1; 8: Buffer B elution 3; 9: Buffer B elution 2; 10: Buffer C elution; 11: Marker. The buffers and elution solutions used were the same as those used in the first heparin column purification. Figure 4 Electrophoresis results showed that the CFE6 fusion protein bound well to the heparin column, the eluted protein was of high purity and small in volume, and the protein concentration was concentrated. The final total eluted protein amount was 122.68 mg, the total protein amount before concentration was 130 mg, the recovery rate was 94.4%, and there was almost no loss of the target protein.

[0087] Example 5: Dialysis and dispensing of CFE6 fusion protein

[0088] Three eluents were dialyzed into a buffer solution of 10 mM PB, 150 mM NaCl, pH 6.5, with a total protein solution volume of 185 ml and a dialysate volume of 5 L. Dialysis was performed overnight, with the dialysate changed once the following day, followed by another 8 hours of dialyzing. The highest protein concentration after dialysis was 60 ml, with an OD of [missing value]. 280 The concentration was 1.01 mg / ml, and the OD value was 1.590. The sample was filtered under sterile conditions using a 0.22 μm microporous membrane. 280 The concentration was 0.83 mg / ml, with a value of 1.299. The solution was aliquoted into 3 ml vials (de-endotoxin treated), 1.2 ml per vial, containing 1 mg per vial, for a total of 48 vials, and stored at -80°C. The remaining protein solutions were stored at -80°C for use in functional studies of FGF1 and Pt.

[0089] Example 6: Agarose gel microspheres with heparin as a ligand were constructed to evaluate the binding affinity of different combinations of FGF1 and Pt fusion proteins to heparin.

[0090] Based on different heparin ligand concentrations, the optimal heparin density on the packing microspheres was determined to be 5 mg heparin / ml microspheres. Under conditions of 20 mM PB, pH 6.8, 1 mM EDTA, and 250 mM NaCl, 100% binding capacity was achieved with either 6.67 mg FGF1 / ml microspheres or 1.33 mg FGF1 / mg heparin. Using this heparin-conjugated microsphere packing material, we conducted binding experiments on different transmembrane peptides and FGF1 fusion proteins (CFE1-CFE8) in different combinations.

[0091] Given that FGF1 is a large molecule with 140 amino acid residues, we first attempted to reduce its molecular weight. Suh (2014) demonstrated that removing the nine N-terminal amino acid residues (FGF1)... ΔNT24 The 131 amino acid residues of FGF1 significantly reduced cell proliferation, but it still retained its complete hypoglycemic effect (Suh JM et al. 2014. Endocrinization of FGF1 Produces a Neomorphic and Potent Insulin Sensitizer. Nature. 513:436–439). Therefore, we selected FGF1. ΔNT24 .

[0092] Meanwhile, we selected two cell-penetrating peptides: 1) M2 (DRFFFKRIYRRLKYRLKRRPST) (Jiang Chengyu et al., 2020, Cell-penetrating peptides based on influenza virus M2 protein, International application number: PCT / CN2019 / 115107, International application date: 2019.11.1, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences); M2 cell-penetrating peptides are newly discovered from influenza virus M2 protein and have been shown to have a strong ability to penetrate cell membranes.

[0093] 2) Penetratin or Pt (RQIKIWFQNRRMKWKK) derived from Drosophila antennal peptide ANTP (Xie Yangyang, Wang Shaojuan, Yuan Quan, Xia Ningshao. 2019. New progress in the research and application of cell membrane-penetrating peptides. Chinese Journal of Biotechnology. 35(7):1162-1173).

[0094] Based on the amino acid sequence of the fusion protein and E. coli codon optimization, we constructed and expressed CFE1(M2-FGF1). ΔNT24 ) and CFE2(Pt-FGF1 ΔNT24 That is, placing M2 or Pt in FGF1 ΔNT24 The N-terminus of the sample was examined, and it was found that both could only bind a very small portion of the heparin column. Figure 5 ), Figure 5In the SDS-PAGE assay, 12% was performed with a loading volume of 5 μl / lane. The results showed that only a small portion of the CFE2 fusion protein could bind to the heparin column, and high-concentration salt (1.5 M NaCl) was required for elution. This indicates that CFE2 not only lacks FGF1 activity but is also difficult to separate and purify. The arrows in the figure indicate the eluted CFE2. Lanes: 1: CFE2 E. coli expression sample; M: Marker; 2: Flow-through; 3-8: Elution with Buffer B to Buffer G, using the following eluents:

[0095] Buffer A: 20mM PB+1mM EDTA (pH7.0)

[0096] Buffer B: 20mM PB+1mM EDTA+100mM NaCl (pH7.0)

[0097] Buffer C: 20mM PB+1mM EDTA+200mM NaCl (pH7.0)

[0098] Buffer D: 20mM PB+1mM EDTA+300mM NaCl (pH7.0)

[0099] Buffer E: 20mM PB+1mM EDTA+500mM NaCl (pH7.0)

[0100] Buffer F: 20mM PB+1mM EDTA+1.5MNaCl (pH7.0)

[0101] Buffer G: 0.1M NaOH.

[0102] Since heparin binding is a necessary condition for FGF1 to exhibit function, it is impossible to have FGF1 function without heparin binding. Therefore, N-terminal fusion of Pt or M2 transmembrane proteins can easily alter the heparin binding capacity and activity of FGF1.

[0103] We further explored using the intact FGF1 (140 amino acid residues) to fuse Pt with FGF1 in three ways: without a linker (CFE3); with a flexible linker (GGGGS)3 (CFE4); and with a rigid linker (A(EAAAK)3A) (CFE5). The results showed that all three fusion proteins could only bind a very small portion of heparin. This demonstrates that fusing Pt with FGF1 at the N-terminus, regardless of whether a flexible, rigid, or without linker is used, cannot yield a fusion protein with good heparin binding.

[0104] Because the N-terminal linker of Pt to FGF1 prevents the fusion protein from effectively binding heparin, we attempted C-terminal fusion. For this purpose, we designed CFE6 (FGF1-Pt), CFE7 [FGF1-(GGGGS)3-Pt], and CFE8 [FGF1-(A(EAAAK)3A)-Pt]. Results in Examples 3-4 show that CFE6 has the highest heparin binding capacity. Our results also showed that CFE8 has a high heparin binding capacity, but CFE7 only binds a small amount. This indicates that the position of Pt in the fusion protein and the presence or absence of a linker peptide both affect the binding of FGF1 to heparin. Our experimental results demonstrate that CFE6, i.e., placing Pt at the C-terminus of FGF1 without adding a linker peptide, has the best heparin binding capacity and also the highest expression level in *E. coli*, providing material for the study of Pt's transmembrane peptide function and how Pt affects the spatial structure and activity of FGF1.

[0105] Seq ID No. 1: Human mature acidic fibroblast growth factor-FGF1:

[0106] D.

[0107] Seq ID No. 2: Amino acid sequence of Drosophila antennal peptide Penetratin (Pt):

[0108] RQIKIWFQNRRMKWKK.

[0109] Seq ID No. 3: Rigidly linked peptide amino acid sequence:

[0110] AEAAAKEAAAKEAAAKA.

[0111] Seq ID No. 4: Flexible linker peptide amino acid sequence:

[0112] GGGGSGGGGSGGGGS.

[0113] Seq ID No. 5: CFE6 (where the first 141 amino acids are FGF1, and the 16 bolded amino acid residues are Pt):

[0114] Seq ID No. 6 is the DNA sequence encoding FGF1 (lowercase text indicates plasmid sequence, underlined). catATG and ctcgag respectively For pET28 restriction sites Nde I and Xho I):

[0115]

[0116] Seq ID No. 7: DNA sequence encoding the Drosophila antennal peptide Penetratin (Pt):

[0117] CGTCAGATTAAAATTTGGTTTCAGAACCGTCGCATGAAATGGAAGAAA.

[0118] Seq ID No. 8: DNA sequence encoding the amino acid sequence of a rigidly linked peptide.

[0119] GGTGGTGGTGGCTCTGGTGGAGGTGGTAGTGGCGGTGGAGGTTCA.

[0120] Seq ID No. 9: DNA sequence encoding the amino acid sequence of a flexible linker peptide:

[0121] GCGGAAGCAGCAGCCAAAGAAGCTGCAGCGAAAGAAGCGGCAGCTAAAGCG.

[0122] Seq ID No. 10: DNA sequence encoding CFE6 (where the first 141 amino acids are FGF1, and the 16 bolded amino acid residues are Pt):

[0123] The two ends are Nde I and Xho I restriction sites, and the thickened sequence is the DNA sequence encoding Pt.

[0124]

[0125] CFE1 (Seq ID No. 11): M2-FGF1 ΔNT24 (154aa)

[0126] M-DRFFFKRIYRRLKYRLKRRPST

[0127] KPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEK NWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD.

[0128] CFE2(Seq ID No.12): Pt-FGF1 ΔNT24 (148aa):

[0129] M-RQIKIWFQNRRMKWKK

[0130] KPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSTPNEECLFLERLEENHYNTYISKKHAEK NWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD。

[0131] CFE3(Seq ID No.13):Pt-FGF1(157aa)

[0132] M RQIKIWFQNRRMKWKK

[0133] FNLPPGNYK KPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAE SVGEVYIKSTETGQYLAMDT DGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR GPRTHYGQKAILFLPLPLPVSS D。

[0134] CFE4(Seq ID No.14):Pt-(GGGGS)3-FGF1(172aa)

[0135] M RQIKIWFQNRRMKWKK

[0136] GGGGSGGGGSGGGGS

[0137] FNLPPGNYK KPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAE SVGEVYIKSTETGQYLAMDTDGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR GPRTHYGQKAILFLPLPLPVSS D。

[0138] CFE5(Seq ID No.15):Pt-A(EAAAK)3A-FGF1(174aa)

[0139] M RQIKIWFQNRRMKWKK

[0140] AEAAAKEAAAKEAAAKA

[0141] D.

[0142] CFE7(Seq ID No.16):FGF1-(GGGGS)3-Pt(172aa)

[0143] M FNLPPGNYK KPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAE SVGEVYIKSTETGQYLAMDTDGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR GPRTHYGQKAILFLPLPVSS DGGGGSGGGGSGGGGS

[0144] RQIKIWFQNRRMKWKK.

[0145] CFE8(Seq ID No.17):FGF1-A(EAAAK)3A-Pt(174aa)

[0146] M FNLPPGNYK KPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAE SVGEVYIKSTETGQYLAMDTDGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR GPRTHYGQKAILFLPLPVSS DAEAAAKEAAAKEAAAKA

[0147] RQIKIWFQNRRMKWKK.

[0148] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fusion protein FGF1-Pt, characterized in that, The amino acid sequence of the fusion protein FGF1-Pt is shown in Seq ID No.

5.

2. A plasmid containing a nucleotide sequence encoding the fusion protein FGF1-Pt as described in claim 1.

3. A host cell, characterized in that, The host cell contains the plasmid as described in claim 2.

4. A method for preparing the fusion protein FGF1-Pt, characterized in that, The encoding DNA of the fusion protein described in claim 1 is inserted into an expression vector, and this vector is introduced into a prokaryotic expression system for expression.

Images