Modular fusion proteins for intracellular protein delivery

Abstract

This invention discloses a modular fusion protein for intracellular protein delivery, which can effectively deliver any target protein into the cytoplasm. The fusion protein consists of multiple functional modules, including a cell-penetrating peptide, a lysosomal localization sequence, a lysosomal responsive cleavage linker peptide, a protein-protein interaction module (such as a nanobody sequence or streptavidin sequence), and a lysosomal escape sequence. The protein-protein interaction module binds to the target protein, which then enters the cell and is transported to the lysosome under the guidance of the cell-penetrating peptide and the lysosomal localization sequence. Subsequently, the lysosomal responsive cleavage linker peptide is specifically cleaved. The target protein bound to the lysosomal escape sequence is effectively delivered into the cytoplasm through the pH-dependent membrane disruption properties of the lysosomal escape sequence. This modular fusion protein enables safe, efficient, and high-uptake cytoplasmic protein delivery, and has broad application prospects in the life sciences and biomedicine fields.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a modular fusion protein and its application in intracellular protein delivery. Background Technology

[0002] Proteins play a crucial role in cell signal transduction, and many genetic diseases are caused by abnormal protein function. Currently, the U.S. Food and Drug Administration has approved over 100 protein drugs and therapies, attracting widespread attention, including insulin, peptide protacs, and antibody-drug conjugates (ADCs). However, these protein drugs are often limited by their ability to penetrate cell membranes, thus limiting their application to intracellular targets. Cellular protein delivery technologies offer a transformative solution for biomedical applications involving intracellular targets. Although techniques for delivering antibodies and other bioactive proteins (such as microinjection and electroporation) have been reported, innovative and practical methods are still needed to achieve adequate interaction with intracellular targets and reach effective levels without cytotoxicity.

[0003] The cell membrane acts as a natural barrier, restricting protein entry into the cytoplasm. Although a few small proteins can enter via receptor-mediated endocytosis, this is only applicable to specific proteins. Furthermore, proteins need to overcome limitations in their release from endosomes into the cytoplasm. Most intracellular delivery strategies rely on modifying proteins with endocytic motifs or nanocarriers, but limiting factors such as vesicle encapsulation and lysosomal pathways can affect delivery efficiency. Designing carriers that trigger lysosomal rupture and escape can improve this situation.

[0004] To address the challenges of intracellular protein delivery, various nanocarriers have been developed, including polymers, inorganic materials, lipid nanoparticles, and engineered viral particles. These carriers can encapsulate and deliver proteins through physical interactions or chemical binding. Furthermore, engineered toxin sequences and other peptide sequences can be used for protein drug delivery. To achieve cytoplasmic delivery independent of endocytosis, proteins or nanosystems can be modified with cell-penetrating peptides, phase-separating peptides, or cholesterol tags, enabling them to cross the cell membrane directly. However, the absorption efficiency of these strategies is often affected by temperature or serum, and some nanomaterials may present toxicity issues. For example, cationic polymers can achieve lysosomal escape through the proton sponge effect. After proton uptake from a lysosome, the pH within the lysosome increases, leading to decreased lysosomal membrane stability. This can trigger lysosomal rupture and release its contents into the cytoplasm, affecting normal cell function. Compared to other delivery strategies, peptide-mediated cytoplasmic protein delivery is a safe, efficient, and high-uptake alternative that can advance intracellular target engagement in drug research. Summary of the Invention

[0005] The purpose of this invention is to design a modular fusion protein that can efficiently deliver any target protein into the cytoplasm. This fusion protein consists of multiple functional modules, including a cell-penetrating peptide, a lysosomal localization sequence, a cleavable lysosomal-responsive linker peptide sequence, a protein-protein interaction module (such as a nanobody sequence or streptavidin MSA sequence), and a lysosomal escape sequence. For ease of in vitro purification, it may also include a protein purification tag (such as a His-tag).

[0006] The modular fusion protein provided in this application comprises, from the N-terminus to the C-terminus, parts A, B, C, D, E, and F, wherein part A is a cell-penetrating peptide, part B is a lysosomal localization sequence, part C is a lysosomal responsive linker peptide, part D is a protein-protein interaction module, part E is a flexible linker peptide, and part F is a lysosomal escape sequence. The protein-protein interaction module is a polypeptide sequence capable of interacting with the target protein to be delivered and binding them together, such as a nanobody sequence, a monomeric streptavidin sequence (MSA), or one of two other interacting protein sequences.

[0007] The modular fusion protein described above is mixed with the target protein to be delivered, and their binding is achieved in vitro through a protein interaction module. For example, the target protein to be delivered is fused with a fluorescent tag and then bound to the modular fusion protein containing a nanobody sequence of the fluorescent tag; or, the target protein to be delivered is biotinylated and then bound to the modular fusion protein containing a streptavidin sequence. Then, guided by the cell-penetrating peptide and lysosomal localization sequence, the modular fusion protein bound to the target protein directly enters the cell and is transported to the lysosome. In the lysosome, the lysosomal response of the fusion protein's cleavage linker peptide is specifically cleaved by an enzyme, separating it into two parts: the lysosomal localization sequence and the target protein portion fused with the lysosomal escape sequence. Subsequently, due to the pH-dependent membrane disruption properties of the lysosomal escape sequence, the target protein is efficiently delivered into the cytoplasm. Notably, the designed fusion protein is localized in the lysosome via the lysosomal localization sequence and intelligently discards non-target protein fragments due to the responsiveness of the lysosome, thereby reducing the burden of cytoplasmic protein delivery. Compared to nanomaterials that disrupt lysosome escape, the fusion protein of this invention does not harm cells, and the remaining proteins in the lysosome will continue to degrade.

[0008] The cell-penetrating peptides in part A mentioned above, such as Pep-1, have the amino acid sequence KETWWETWWTEWSQPKKKRKV (SEQ ID No: 1 in the sequence listing). In addition to Pep-1 cell-penetrating peptides, part A can also be, but is not limited to, cationic, amphiphilic and hydrophobic penetrating peptides, such as TAT, MAP, Transportan, MPG, Bip, Pep-7, FGF, etc.

[0009] The lysosomal localization sequences in part B mentioned above, such as saposin A, have the amino acid sequence GSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCES (SEQ ID No: 2 in the sequence listing); other lysosomal localization sequences include, but are not limited to, saposin B, saposin C, saposin D, lamp1, lamp2, and lamp3.

[0010] The lysosomal response cleavage linker peptide in part C is preferably of the amino acid sequence GGFG, which can be specifically cleaved by Cathepsin B.

[0011] The aforementioned flexible linker peptide in part E is preferably (GGGGS). n , where n is an integer from 1 to 4.

[0012] The lysosomal escape sequence of the F portion is preferably selected from the GALA polypeptide sequence (SEQ ID No: 4-9 in the sequence listing) and related truncated sequences, such as the GALA, GALA-1 to GALA-5 sequences shown in the table below.

[0013] sequence name amino acid sequence GALA WEAALA EALAEALAEH LAEALA EALEALAA GALA-1 WEAALA GALA-2 WEAALA EALEALAA GALA-3 LAEALA EALEALAA GALA-4 EALAEALAEH LAEALA EALEALAA GALA-5 EALEALAA

[0014] Furthermore, to facilitate in vitro purification, the modular fusion protein also has a G portion at the C-terminus—a protein purification tag, such as a histidine tag (His-tag) sequence.

[0015] The aforementioned D-part protein-protein interaction module, when it is a nanobody sequence, such as an mCherry nanobody (the target protein to be delivered is fused with an mCherry tag), was used in embodiments of the present invention to prepare fusion proteins PSMG and PSMG3 with the following sequences:

[0016] PSMG fusion protein sequence:

[0017] MKETWWETWWTEWSQPKKKRKVMGSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESGGFG SAQVQLVESGGGLVQAGGSLLRLSCATSGFTFSDYA MGWFRQAPGKEREFVAAISWSGHVTDYADSVKGRFTISRDNVKNTVYLQMNSLKPEDTAVYSCAAAKSGTWWYQRS ENDFGSWGQGTQVTVS GGGGSGGGGSWEAALAEALAEALAEHLAEALAEALEALAAHHHHHH (SEQ ID No: 10 in the sequence listing, the underlined part is the nanobody sequence).

[0018] PSMG3 fusion protein sequence:

[0019] MKETWWETWWTEWSQPKKKRKVMGSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESGGFG SAQVQLVESGGGLVQAGGSLLRLSCATSGFTFSDYA MGWFRQAPGKEREFVAAISWSGHVTDYADSVKGRFTISRDNVKNTVYLQMNSLKPEDTAVYSCAAAKSGTWWYQRS ENDFGSWGQGTQVTVS GGGGSGGGGSLAEALAEALEALAAHHHHHH (SEQ ID No: 11 in the sequence listing, the underlined part is the nanobody sequence).

[0020] In addition to the mcherry nanobody sequence, the D part of the modular functional protein can also be other nanobody sequences, such as GFP nanobody, Flag nanobody, etc., with the target protein to be delivered having the corresponding protein tag GFP, Flag, etc.

[0021] When part D is a monomeric streptavidin sequence (the target protein to be delivered is biotinylated), fusion proteins PSSG and PSSG3 with the following sequences were prepared:

[0022] PSSG fusion protein sequence:

[0023] MKETWWETWWTEWSQPKKKRKVMGSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESGGFG AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRA QGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFT KVK GGGGSGGGGSWEAALAEALAEALAEHLAEALAEALEALAAHHHHHH (SEQ ID No: 12 in the sequence listing, the underlined part is the streptavidin sequence).

[0024] PSSG3 fusion protein sequence:

[0025] MKETWWETWWTEWSQPKKKRKVMGSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESGGFG AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRA QGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFT KVK GGGGSGGGGSLAEALAEALEALAAHHHHHH (SEQ ID No: 13 in the sequence listing, the underlined part is the streptavidin sequence).

[0026] The encoding gene and expression cassette of the modular fusion protein, the genetic engineering vector containing the encoding gene of the modular fusion protein, the host bacteria containing the genetic engineering vector, and the kit containing the modular fusion protein are all within the scope of protection of this invention.

[0027] By employing fusion expression (e.g., fusing an mCherry tag with the target protein to be delivered, followed by incubation and mixing with a fusion protein containing an mCherry nanobody) or direct mixing (the target protein to be delivered is biotinylated and then incubated and mixed with a fusion protein containing streptavidin), this invention has successfully achieved efficient intracellular delivery of functional protein drugs both in vivo and in vitro. The fusion protein retains its biological activity and performs its unique application function in the cytoplasm. Based on this hitch-and-run strategy, this invention designs modular fusion proteins to achieve safe, efficient, and high-uptake cytoplasmic protein delivery with potential applications in various cell types. It allows for the flexible application of any nanobody with cargo combinations and facilitates the easy acquisition of fusion proteins through large-scale, consistent protein purification operations. Therefore, the modular fusion proteins of this invention have broad application prospects in the life sciences and biomedical fields. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the fusion protein sequence and its different components used for delivering proteins into cells according to the present invention.

[0029] Figure 2 This is a schematic diagram of protein binding, where: A shows the superimposed structure of mCherry and LaM nanobody, with the structure from the PDB database superimposed onto mCherry (PDB ID: 2H5Q) using Pymol software; B shows the detailed binding sites of LaM2 and mCherry, with key residues in LaM2 used for binding mCherry shown in the figure.

[0030] Figure 3 The predicted structure diagram of the PSMG and mCherry complex is shown.

[0031] Figure 4 The binding affinity between PSMG and DUF5-mCherry is measured by ITC, and the binding constant (KD value ± standard deviation) and stoichiometry (N) are given. The KD value and standard deviation were calculated from three independent experiments.

[0032] Figure 5 To verify that PSMG can deliver proteins of different sizes (mCherry: 26.7KD and DUF5-mCherry: 79.4KD) into cells, cytoplasmic fluorescence was observed using laser confocal microscopy.

[0033] Figure 6 To observe the distribution of fluorescence in different cell lines (B16 cells and HeLa cells) using laser confocal microscopy to deliver mCherry protein via PSMG (incubated at 37°C for 16 h).

[0034] Figure 7 To observe the distribution of fluorescence in different cell lines (PANC1 and HEK-293) using laser confocal microscopy to deliver mCherry protein via PSMG (incubated at 37°C for 16 h).

[0035] Figure 8 To deliver mCherry protein in SF9 cells using PSMG, cells were incubated at 37°C for different times (0, 0.5, 1, 2 h), and the distribution of fluorescence in the cells (nuclear blue fluorescent dye Hoechst 33342) was observed using laser confocal microscopy.

[0036] Figure 9 To deliver mCherry protein in HeLa cells using PSMG, cells were incubated at 37°C for different times (0, 2, 6, 8, 10 h), and the distribution of fluorescence in the cells (nuclear blue fluorescent dye Hoechst 33342) was observed using laser confocal microscopy.

[0037] Figure 10 To investigate the pathway of mCherry protein delivery by PSMG, we treated HeLa cells with different inhibitors of different entry pathways and then used flow cytometry to detect the entry pathway.

[0038] Figure 11 To detect the cell entry efficiency of PSMG-delivered mCherry protein in HeLa cells (under culture conditions with 10% FBS serum) by flow cytometry.

[0039] Figure 12To investigate whether the GALA sequence in the fusion protein could play a role in lysosomal escape, laser confocal microscopy was used to detect the fluorescence localization of different proteins in 293 cells (Pep1-SAP-GFP and FITC-modified Pep-SAP-GALA).

[0040] Figure 13 The results of cytotoxicity assays (incubation at 37°C for 6 h) of the fusion protein PSMG at different concentrations (5, 10, 25, 50, 100 μg / mL) in different cell lines were obtained.

[0041] Figure 14 The results of the hemolysis experiment in Example 14 are as follows: A. The results of the in vitro safety evaluation of PSMG at different concentrations (10, 25, 50, 100, 200 μg / mL) using the hemolysis experiment, where NC is the negative control phosphate buffer solution and PC is the positive control distilled water; B. The statistical results of the hemolysis rate of the PSMG fusion protein treatment groups at different concentrations in the hemolysis experiment.

[0042] Figure 15 To deliver mCherry protein in HeLa cells using PSSG3, the cells were incubated at 37°C for 3 hours, and the distribution of fluorescence in the cells was observed using laser confocal microscopy. Detailed Implementation

[0043] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0044] Example 1: Purification of PSMG protein

[0045] An expression vector pET-28a(+)-PSMG for the PSMG protein, as shown in SEQ ID No: 10, was constructed. The plasmid derived from the pET-28a(+)-PSMG vector was transformed into *E. coli* BL21(DE3) cells and incubated overnight at 37°C (OD value 0.8). Protein expression was then induced for 15 hours at 20°C with 1 mM IPTG (final concentration 0.1 mM). The bacterial pellet was harvested and resuspended in lysis buffer containing 20 mM HEPES buffer (pH 7.4) and 150 mM NaCl. The supernatant was collected by sonication (using a cell sonicator for 10 minutes at 150 W) and centrifugation at 12000 rpm for 20 minutes. Subsequently, the His-tagged PSMG protein was purified using a Ni-IDA gravity chromatography column, and further purified protein separation and collection were performed using a protein purifier. Protein concentration was measured using a NanoDrop protein concentration analyzer, and the contents were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C until use.

[0046] Example 2: Analysis of the superposition structure of mCherry with different mCherry nanobodies (LaM)

[0047] Due to their small size and strong binding affinity, nanobodies are ideal for carrying target proteins. Among numerous fluorescent proteins, mCherry is the most widely used. This is because mCherry exhibits several desirable properties, including high brightness, excellent photostability, and emission wavelengths suitable for imaging applications. Most importantly, its monomeric nature ensures that it does not form tetramers; fusing mCherry protein with the target protein does not affect the target protein's function and reduces the possibility of false positive binding results. Therefore, we chose mCherry nanobodies to carry the target protein into cells.

[0048] Currently, research on mCherry nanobodies is quite mature, including LaM1-8. We downloaded the complex files of these nanobodies and mCherry proteins from the PDB protein database. After opening them with PyMOL software, we compared these nanobodie subtypes. Figure 2As shown in Figure A, the binding states of nanobodies to mCherry are illustrated from different perspectives. Since target proteins are typically fused to the N-terminus of mCherry, binding the nanobodies to the β-barrel of mCherry in our fusion protein would minimize the possibility of steric hindrance. Because the LaM4 recognition site is located at the N-terminus and C-terminus of mCherry, LaM4 is excluded. Other nanobodies, LaM1, LaM2, LaM3, LaM6, and LaM8, can all bind to the β-barrel structure of mCherry. Among them, the β-barrels of mCherry and LaM2 stand out due to their high binding affinity of 3.02 nM KD. Structural analysis reveals, as... Figure 2 The detailed binding sites of LaM2 and mCherry are shown in Figure B: The key residues for the binding of the nanobody LaM2 to mCherry are Trp67, Trp119, and Tyr120, which are located far from the N and C ends of LaM2, and the fusion of the protein at its N and C ends does not interfere with the interaction between mCherry and LaM2.

[0049] Example 3: Predicted structure of the PSMG and mCherry complex using Alphabold2

[0050] We performed protein complex structure prediction on the designed fusion protein PSMG (containing the LaM2 nanobody sequence) for delivery and the mCherry protein. Data from Alphabold2 validated the conclusions of Example 2, as shown below. Figure 3 The results show that PSMG and mCherry form a sterically unhindered complex. From a structural analysis perspective, PSMG shows great promise in binding to target proteins fused with mCherry.

[0051] Example 4: ITC measurement of binding affinity between PSMG and DUF5-mCherry

[0052] To verify the binding affinity of PSMG to DUF5-mCherry (a fusion protein of DUF5 and mCherry), we further used isothermal titration calorimetry (ITC) to measure the binding affinity between PSMG and the DUF5-mCherry fusion protein, which showed a binding affinity of 40 nM. The isothermal titration calorimetry (ITC) results (see...) Figure 4The results show that the LaM2 nanobody, after being fused into PSMG, still maintains a strong affinity for the DUF5-mCherry fusion protein, indicating that fusing mCherry with DUF5 does not interfere with the binding of LaM2 to mCherry. These results demonstrate that our structure-information-based strategy is effective for mCherry-labeled proteins.

[0053] Example 5: Verifying the ability of PSMG to deliver proteins

[0054] To verify that PSMG release can deliver proteins into cells, we used confocal microscopy to observe the effects of different proteins (mCherry: 26.7 KD and DUF5-mCherry: 79.4 KD) on cell entry. HeLa cells (5 × 10⁻⁶) were used. 5 Cells were cultured overnight in confocal microscopy dishes, and then different proteins were added: mCherry, PSMG@mCherry, DTM (DUF5-mCherry), and PSMG@DTM. Before adding the proteins to the confocal microscopy dishes, the proteins to be delivered (mCherry and DTM) were mixed separately with PSMG at a molar ratio of 1:1, allowed to stand at room temperature for five minutes, and then added to the confocal microscopy dishes. The mixtures were incubated at 37°C for 4 hours, and then the red fluorescence channel and bright field were observed using a confocal microscope to observe the cell entry of different proteins. Figure 5 As shown, mCherry protein generally cannot actively enter cells; only a portion appears on the cell membrane or inside the cell. This phenomenon may be due to small proteins entering cells via endocytosis mediated by membrane surface receptors. After delivery using PSMG, significant red fluorescence appeared in the cells, indicating that PSMG can deliver mCherry protein into the cells. DTM protein, after incubation with cells, could not enter the cells and showed no fluorescence. This indirectly confirms that large proteins cannot passively enter cells via membrane surface receptors. However, with the assistance of PSMG, the PSMG@DTM group showed full-field red fluorescence. These results demonstrate that PSMG protein has the potential to deliver proteins into cells.

[0055] Example 6: PSMG delivery of mCherry protein in different cell lines (B16 cells and HeLa cells)

[0056] mCherry protein was delivered using PSMG in different cell lines (B16 cells and HeLa cells), incubated at 37°C for 16 h, and the protein concentration was 200 μg / mL. The distribution of fluorescence in the cells was observed using laser confocal microscopy. Green fluorescence was the cell membrane dye WGA-FITC, red fluorescence was the fluorescence of the delivered mCherry protein, and blue fluorescence was the nuclear dye Hoechst 33342. Figure 6As shown, based on previous experimental experience, after adding the PSMG and mCherry complex to cells and incubating for 16 hours, full-field red fluorescence appeared in the cytoplasm, indicating that PSMG can effectively deliver the target protein mCherry into the cytoplasm.

[0057] Example 7: PSMG was used to deliver mCherry protein in different cell lines (PANC1 cells and HEK-293 cells) (incubated at 37°C for 16 h), and the distribution of fluorescence in the cells was observed using laser confocal microscopy.

[0058] mCherry protein was delivered using PSMG in different cell lines (PANC-1 and HEK293T cells), incubated at 37°C for 16 h, and the protein concentration was 200 μg / mL. The distribution of fluorescence in the cells was observed using laser confocal microscopy. Green fluorescence is the cell membrane dye WGA-FITC, and red fluorescence is the fluorescence of the delivered mCherry protein. Figure 7 As shown, a full field of red fluorescence appeared in the cytoplasm, which verifies the results in Example 6, indicating that PSMG can effectively deliver the target protein mCherry into the cytoplasm.

[0059] Example 8: PSMG was used to deliver mCherry protein in SF9 cells (with different incubation times) and the distribution of fluorescence in the cells was observed using laser confocal microscopy.

[0060] SF9 cells were cultured in confocal microplates, and then a complex of PSMG and mCherry (molar ratio 1:1) was added. The concentration of mCherry was 100 μg / mL. The cells were then incubated at 37°C for different times (0, 0.5, 1, 2 h). The distribution of fluorescence in the cells was observed using laser confocal microscopy (nuclear staining was done with the blue fluorescent dye Hoechst 33342).

[0061] The results are as follows Figure 8 As shown, a large amount of red fluorescence appeared in SF9 cells over time, indicating that PSMG can effectively deliver mCherry protein into SF9 cells.

[0062] Example 9: PSMG was used to deliver mCherry protein in HeLa cells (with different incubation times) and the distribution of fluorescence in the cells was observed using laser confocal microscopy.

[0063] A complex of PSMG and mCherry (molar ratio 1:1, mCherry protein concentration 100 μg / mL) was added to HeLa cells and incubated at 37°C for different times (0h, 2h, 6h, 8h, 10h). The distribution of fluorescence in the cells was observed using laser confocal microscopy. Green represents the lysosomal probe, and red represents the fluorescence of the delivered mCherry protein.

[0064] like Figure 9 As shown, the red fluorescence in the cells increased with prolonged incubation time, indicating that PSMG can deliver proteins into the cells. During 10 hours of incubation, most of the proteins delivered by PSMG remained within lysosomes. Comparing different incubation times, 16 hours and longer incubation times in HeLa cells are more suitable for PSMG to deliver proteins into the cytoplasm.

[0065] Example 10: Detection of the cell entry pathway of the PSMG@mCherry protein complex using different cell entry inhibitors

[0066] To investigate the pathway by which PSMG@mCherry enters cells, we pretreated HeLa cells for 1 h with different cell pathway inhibitors (chlorpromazine CPZ, genistein, methyl-β-cyclodextrin MβCD, nystatin, and amiloride). Then, we added the PSMG@mCherry protein complex (mCherry concentration: 100 μg / mL) and incubated at 37°C for 2 h. After incubation with the commercially available lysosomal probe Lyso-Trackerred (concentration: 10 μmol / L) for 10 min, washed three times with phosphate-buffered saline (PBS), and observed and photographed using laser confocal microscopy. Methyl-β-cyclodextrin significantly reduced clathrin-dependent endocytosis; amiloride hydrochloride inhibited endocytosis via macropinocytosis; and nystatin and genistein both inhibited endocytosis via cell membrane pits.

[0067] Stream cytometry statistics as follows Figure 10 The results showed that the fluorescence intensity statistics in the above groups were basically consistent with those in the control group, indicating that PSMG is not limited by conventional inhibitors of cell entry pathways.

[0068] Example 11: Investigating the effect of serum on cellular uptake of the PSMG@mCherry complex

[0069] HeLa cells were cultured in six-well plates (complete culture medium containing 10% fetal bovine serum). PSMG@mCherry complex (molar ratio 1:1, mCherry protein concentration 100 μg / mL) was added to the wells. After incubation for 6 h, the fluorescence intensity of mCherry was detected by flow cytometry and statistically analyzed.

[0070] Streaming results as follows Figure 11 As shown, when the PSMG@mCherry protein complex was co-incubated with HeLa cells containing serum, the fluorescence intensities were 99.4%, 99.6%, and 99.7%, respectively, indicating that the HeLa cells almost completely took up the PSMG@mCherry protein. This demonstrates that the cellular uptake of the PSMG@mCherry complex is not affected by the presence of serum.

[0071] Example 12: Detecting whether the GALA sequence in the fusion protein can play a role in lysosomal escape.

[0072] To evaluate the feasibility of lysosomal escape sequences facilitating peptide release from lysosomes into the cytoplasm, we validated this in 293 cells using Pep1-SAP-GFP protein (fused with GFP protein for fluorescent tracking) and Pep1-SAP-GALA (labeled with FITC green fluorescent dye). Here, Pep1 represents the cell-penetrating peptide Pep-1, SAP represents the lysosomal localization sequence saposin A, and GALA represents the lysosomal escape sequence. The amino acid sequence of the fusion protein Pep1-SAP-GALA constructed in this example is as follows:

[0073] MKETWWETWWTEWSQPKKKRKVMGSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESGGGGSGGGGSWEAALAEALAEALAEHLAEALAEALEALAAHHHHHH (SEQ ID No: 14 in the sequence listing).

[0074] The above proteins (concentration: 100 μg / mL) were added to the cells, and after incubation at 37°C for 4 h, the cells were incubated with commercially available red lysosomal probe Lyso-Tracker red (concentration 10 μmol / L) for 10 min. After washing three times with phosphate-buffered saline (PBS), laser confocal microscopy was performed for observation and imaging.

[0075] Laser confocal results are as follows Figure 12The results showed that the green fluorescence of the Pep1-SAP-GFP protein co-localized with the red fluorescence of the lysosomal probe, indicating that the Pep1-SAP-GFP protein is mainly located in the lysosomes of the cell. The green fluorescence of Pep1-SAP-GALA (labeled with FITC green fluorescent dye) was diffusely distributed in the cytoplasm, indicating that it had escaped from the lysosomes into the cytoplasm.

[0076] Example 13: Cytotoxicity assay of the fusion protein in different cell lines

[0077] To evaluate the safety of the fusion protein, we used PSMG protein as an example and added different concentrations of PSMG protein (5, 10, 25, 50, and 100 μg / mL) to different cell lines HEK293T (human embryonic kidney cells), B16 (melanoma cell line), and HeLa (cervical cancer cell line) for cytotoxicity experiments. Cells were seeded in 96-well plates (cell density: 10,000 cells / well), and after incubation at 37°C for 24 h, different concentrations of PSMG protein were added. After incubation at 37°C for 6 h, the cytotoxicity assay was performed according to the CCK-8 cytotoxicity assay kit at a wavelength of 450 nm.

[0078] Experimental results are as follows Figure 13 The results showed that all three cell types exhibited good growth at different drug concentrations, with a survival rate close to 100%, indicating that the PSMG protein has good biocompatibility.

[0079] Example 14: In vitro safety evaluation results of PSMG at different concentrations (10, 25, 50, 100, 200 μg / mL) using a hemolysis test.

[0080] Hemolysis assays are typically used to assess the destructive effects of substances on erythrocytes when exposed to them. Take 1 mL of rabbit erythrocytes in a 15 mL centrifuge tube, add 9 mL of phosphate-buffered saline (PBS, pH 7.4), mix well, and centrifuge at 1500 rpm for 5 min. Discard the supernatant and add PBS. Repeat this process three times. Take 1 mL of erythrocytes from the bottom and add 9 mL of PBS to prepare a 10% erythrocyte solution. Use the 10% erythrocyte solution for sample preparation: set up a positive control group, a negative control group, and different concentrations of PSMB (concentrations: 10, 25, 50, 100, 200 μg / mL). NC is the negative control: PBS; PC is the positive control: distilled water. Aliquot each group of samples into EP tubes, incubate at 37°C for 1 h, centrifuge at 3500 rpm for 5 min, and record the hemolysis status of each group. In addition, 0.1 mL of each sample was added to a 96-well plate and the absorbance of the sample was detected using an ELISA reader (detection wavelength: 540 nm), and the hemolysis rate was statistically analyzed.

[0081]

[0082] The results are as follows Figure 14 As shown in Figure A, the in vitro safety evaluation results of PSMG at different concentrations (10, 25, 50, 100, 200 μg / mL) were obtained using a hemolysis assay. NC was the negative control (phosphate buffered solution), and PC was the positive control (distilled water). Figure B shows the statistical results of the hemolysis rate of the PSMG fusion protein at different concentrations in the hemolysis assay. The positive control group PC showed complete hemolysis, while the negative control group NC showed no hemolysis. The hemolysis rates for PSMG protein concentrations of 10, 25, 50, 100, and 200 μg / mL were 0.85%, 1.74%, 1.79%, 2.52%, and 3.08%, respectively. All PSMG concentrations showed low hemolysis rates, all below 5% (ISO standard: hemolysis rate <5%), indicating that PSMG has good blood compatibility.

[0083] Example 15: Detection of cellular entry of PSSG3-delivered biotinylated mCherry protein

[0084] To verify the feasibility of delivering biotinylated proteins to cells using modular proteins containing the monomeric streptavidin sequence (MSA), the protein to be delivered was biotinylated: (+)-biotin-N-hydroxysuccinimide ester was dissolved in DMSO / water solution, then mixed with mCherry protein at a molar ratio of 1:1, and incubated at room temperature for 30 min. The biotinylated mCherry protein was then combined with the modular fusion protein containing the streptavidin sequence, and further purified using a protein purifier before use. In HeLa cells, mCherry protein was delivered using PSSG3 (molar ratio 1:1, mCherry protein concentration 100 μg / mL), incubated at 37 °C for 3 h, and the fluorescence distribution in the cells was observed using laser confocal microscopy (scale bar 75 μm).

[0085] The results are as follows Figure 15 As shown, red fluorescence is diffusely distributed in HeLa cells, indicating that PSSG3 can effectively deliver biotinylated mCherry protein into the cell.

Claims

1. A modular fusion protein, characterized in that, The sequence from the N-terminus to the C-terminus comprises parts A, B, C, D, E, and F. Part A is a cell-penetrating peptide, part B is a lysosomal localization sequence, part C is a lysosomal responsive cleavage linker peptide, part D is a protein-protein interaction module, part E is a flexible linker peptide, and part F is a lysosomal escape sequence. The protein-protein interaction module is a polypeptide sequence capable of interacting with the target protein. Part A is Pep-1, and its amino acid sequence is SEQ ID No:

1. Part B is saposin A, and its amino acid sequence is SEQ ID No:

1. No: 2; Part C is the lysosomal response cleavage linker peptide GGFG; Part D sequence is SAQVQLVESGGGLVQAGGSLRLSCATSGFTFSDYAMGWFRQAPGKEREFVAAISWSGHVTDYADSVKGRFTISRDNVKNTVYLQMNSLKPEDTAVYSCAAAKSGTWWYQRSENDFGSWGQGTQVTVS or AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVK; Part F is SEQ ID No: 4 or SEQ ID No:

7.

2. The modular fusion protein as described in claim 1, characterized in that, The flexible linker peptide in part E is (GGGGS). n , where n is an integer from 1 to 4.

3. A modular fusion protein, characterized in that, The amino acid sequence of the modular fusion protein is selected from one of SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, and SEQ ID No: 13 in the sequence listing.

4. A method for intracellular protein delivery for purposes other than disease diagnosis and treatment, comprising combining a target protein to be delivered with a modular fusion protein as described in any one of claims 1 to 3, wherein the modular fusion protein delivers the target protein into the cell, wherein the D portion of the modular fusion protein is an mCherry nanobody sequence, and the target protein to be delivered is fused with a corresponding mCherry protein tag; or, the D portion of the modular fusion protein is a monomeric streptavidin sequence, and the target protein to be delivered is biotinylated.

Images