A transmembrane fluorescent protein, a water-soluble fluorescent protein and uses thereof

Abstract

The present invention discloses a transmembrane fluorescent protein, a water-soluble fluorescent protein and uses thereof. The amino acid sequence of the transmembrane fluorescent protein is selected from: SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, and the amino acid sequence of the water-soluble fluorescent protein is selected from: SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9. The transmembrane fluorescent protein and the water-soluble fluorescent protein of the present invention can bind to HBC derivatives—HBC599 and HBC620 with high affinity and high specificity, and can be used for fluorescence imaging in cells and on cell membranes.

Description

Technical Field

[0001] The present application belongs to the field of proteins, and specifically relates to a transmembrane fluorescent protein, a water-soluble fluorescent protein and uses thereof. Background Art

[0002] The recognition of small molecules by transmembrane proteins is crucial for the exchange of matter, energy, and information across the cell membrane. There have been many attempts to design transmembrane proteins from scratch to understand the basic principles of small molecule-protein interactions within the cell membrane, thereby achieving tight and specific recognition of specific small molecules; however, progress in this area has been limited to date. There are currently no transmembrane fluorescent proteins designed from scratch.

[0003] All known naturally occurring transmembrane fluorescent proteins are very weak. For the most widely used natural transmembrane fluorescent protein rhodopsin, their fluorescence spectra are limited to their fluorescent group retinal, and their protein topology is limited to 7 transmembrane α-helices. These shortcomings limit their applications.

[0004] Therefore, there is still a need for a new type of transmembrane protein that can bind to fluorescent groups specifically and with high affinity in the cell membrane with high brightness and quantum yield. Such a protein can become a very good tool for sensing, imaging and transmembrane transport on the cell membrane of living cells. Summary of the invention

[0005] The technical problem of the present invention is to provide a class of transmembrane fluorescent proteins and their corresponding water-soluble fluorescent proteins. They bind with high affinity and high specificity to HBC derivatives with fluorescent groups, such as HBC599 and HBC620 (HBC: (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl-acetonitrile, which is a fluorescent group used to bind to RNApepper and has multiple derivatives to produce fluorescence of various wavelengths. Chen X et al., Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotechnol. 2019 Nov; 37(11): 1287-1293.), and their brightness and quantum yield are higher than EGFP, far exceeding all natural fluorescent proteins. These transmembrane proteins can be expressed on the cell membrane of living Escherichia coli (E. coli) and mammalian cells (such as HEK293) for cell membrane sensing, imaging or transmembrane transport.

[0006] On the one hand, the present invention provides a transmembrane fluorescent protein, which includes the amino acid sequence shown in SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, SEQ ID No.:4 or SEQ ID No.:5, or the amino acid sequence of the transmembrane fluorescent protein is SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, SEQ ID No.:4 or SEQ ID No.:5.

[0007] On the other hand, the present invention provides a water-soluble fluorescent protein corresponding to the above-mentioned transmembrane fluorescent protein, wherein the water-soluble fluorescent protein comprises an amino acid sequence shown in SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9, or the amino acid sequence of the water-soluble fluorescent protein is SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9.

[0008] It should be noted that the specificity of the fluorescent proteins of the present invention mainly refers to the fact that these fluorescent proteins specifically bind to the two derivatives HBC599 and HBC620 and excite fluorescence, while the binding and fluorescence excitation with other derivatives such as HBC495, HBC508, and HBC530 are very weak or non-binding.

[0009] The term "corresponding to the above transmembrane fluorescent protein" means that the water-soluble fluorescent protein and the transmembrane fluorescent protein have the same core region that binds to the corresponding HBC molecule. In other words, the ability of these proteins of the present application to bind to the corresponding HBC molecule depends on the amino acid sequence of the core region of the protein, and the sequence of the core region is completely consistent or almost completely consistent. Therefore, the conditions required for the above two types of proteins of the present application to activate fluorescence and the fluorescence spectra after binding to HBC599 / 620 are also very similar, and the core region (see Figure 7 The sequence outside the mutant site shown) mainly determines whether the protein is expressed in the water-soluble environment (water-soluble protein) or the transmembrane environment (transmembrane protein) in the cell.

[0010] On the other hand, the present application provides a polynucleotide encoding the above-mentioned transmembrane fluorescent protein or the above-mentioned water-soluble fluorescent protein.

[0011] In still another aspect, the present application provides an expression vector comprising the above-mentioned polynucleotide.

[0012] On the other hand, the present application provides a cell, which comprises the above-mentioned expression vector, or expresses the above-mentioned transmembrane fluorescent protein, or the above-mentioned water-soluble fluorescent protein.

[0013] In a specific embodiment, the cells include but are not limited to Escherichia coli cells and human HEK293 cells.

[0014] In yet another aspect, the present application provides the use of the above-mentioned transmembrane fluorescent protein, or its expression vector in imaging and sensing on the cell membrane.

[0015] In yet another aspect, the present application provides the use of the above-mentioned water-soluble fluorescent protein, or its expression vector in cell imaging and sensing.

[0016] In yet another aspect, the present invention provides a method for cell membrane imaging, the method comprising using the above-mentioned transmembrane fluorescent protein.

[0017] In yet another aspect, the present invention provides a method for cell imaging, the method comprising using a water-soluble fluorescent protein.

[0018] Technical Effects

[0019] The present application relates to a transmembrane fluorescent protein designed from scratch using a deep neural network, and a corresponding water-soluble fluorescent protein. The transmembrane fluorescent protein and the water-soluble fluorescent protein of the present application bind to HBC derivatives (HBC599 and HBC620) with fluorescent groups with high affinity and high specificity, and their brightness and quantum yield are higher than EGFP, far exceeding all natural fluorescent proteins.

[0020] After the transmembrane fluorescent protein and water-soluble fluorescent protein in the present invention are expressed and purified in Escherichia coli, it can be detected that they can specifically bind to HBC599 and HBC620 molecules. When combined with the corresponding HBC (such as HBC599), the transmembrane fluorescent protein can increase the brightness of HBC by more than 1675 times, the affinity reaches 17nM, the fluorescence quantum yield is 0.87, the shortest fluorescence emission peak is 555nm, and the longest is 590nm; the water-soluble fluorescent protein can increase the brightness of HBC by more than 1150 times, the affinity reaches 51nM, the fluorescence quantum yield reaches 0.88, the shortest fluorescence emission peak is 545nm, and the longest is 590nm.

[0021] These transmembrane fluorescent proteins of the present application can be expressed on the cell membrane of living Escherichia coli (coli) and mammalian cells (such as HEK293) for cell membrane sensing, imaging or transmembrane transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: The amino acid sequence of the water-soluble fluorescent protein (a) and transmembrane fluorescent protein (b) of the present application and the molecular structure of the HBC derivative (c). All wFAPs shown in the figure are mutants modified based on wFAP0, and all tmFAPs are mutants modified based on tmFAP0.1. In the figure, the black dots represent that the sequence of the corresponding position of the mutant has not changed relative to wFAP0 or tmFAP0.1. Red, blue and green are to indicate the positions where the sequence has changed when the mutant is modified. At the positions marked in red or blue, the sequence of the mutant is different from wFAP0 or tmFAP0.1. At the positions marked in green, the sequences of tmFAP2.1 and tmFAP0.1 are different. The positions marked in yellow are the positions where the sequence changes when the water-soluble fluorescent protein is transformed into the transmembrane fluorescent protein.

[0023] Figure 2 :The fluorescence properties of each transmembrane fluorescent protein and water-soluble fluorescent protein of the present application and comparison with natural fluorescent proteins.

[0024] Figure 3 :Detection of the fluorescence properties of the water-soluble fluorescent protein of the present application. (a) Schematic diagram of water-soluble fluorescent protein stimulating HBC599 fluorescence, (b) specificity of water-soluble fluorescent protein, (c) fluorescence spectrum, (d) dissociation constant determination curve of each mutant, (e) luminescence photo display after the protein binds to the HBC derivative, (f) dissociation constant determination curve of wFAP1.1 and different HBC derivatives.

[0025] Figure 4 :Detection of the fluorescence properties of the transmembrane fluorescent protein of the present application. (a) Schematic diagram of the transformation of water-soluble fluorescent protein into transmembrane fluorescent protein, (b) specificity of transmembrane fluorescent protein, (c) fluorescence spectrum, (d) dissociation constant determination curve of each mutant, (e) luminescence photo display after the protein binds to HBC derivatives, (f) dissociation constant determination curve of tmFAP1.2 and different HBC derivatives.

[0026] Figure 5 : Crystal structures of the water-soluble fluorescent protein wFAP1.1 of the present application in two states: bound to HBC (b) and unbound (a). The structure of the interaction between the protein core region and HBC and the comparison with the designed model (c, d).

[0027] Figure 6: Fluorescence photos of the transmembrane fluorescent protein and water-soluble fluorescent protein of the present application after being expressed in living cells (Escherichia coli (a), human HEK293 cells (b)). In Figure a: there is no fluorescence when the protein of the present application is not expressed in Escherichia coli (row 1), the entire cell is fluorescent when the water-soluble fluorescent protein is expressed (row 2), and there is fluorescence on the cell membrane when the transmembrane fluorescent protein is expressed (rows 3 and 4, where row 4 is a partial enlargement of row 3). In Figure b, when the water-soluble fluorescent protein is expressed in human HEK293 cells, the entire cell is fluorescent (row 1), when the mitochondrial localization signal is added, there is fluorescence at the mitochondrial position (row 2), and when the transmembrane fluorescent protein is expressed, there is fluorescence on the cell membrane (rows 3 and 4, where row 4 is a partial enlargement of row 3).

[0028] Figure 7 : Effects of introducing mutations into the binding pocket of wFAP1.1 (upper panel) and tmFAP1.1 (lower panel) on the fluorescence activation signal. DETAILED DESCRIPTION

[0029] The technical solution of the present application is described in detail below through specific embodiments, so that those skilled in the art can better understand the present invention. However, these embodiments are not intended to limit the scope of the present application.

[0030] Example 1: Preparation and purification of water-soluble fluorescent protein

[0031] Water-soluble fluorescent proteins (wFAPs) were constructed using pET-29b vector (with a 6*His tag at the C-terminus of the protein). The plasmid was transferred into E. coli cells (Lemo21(DE3)Competent E.coli, NEB) for expression and cultured in LB medium at 37°C and 220RPM. After the OD reached 0.6, 0.2mM IPTG was added and cultured for 16 hours. wFAPs were expressed by Ni 2+ -NTA affinity chromatography followed by size exclusion chromatography purification. The specific operation is as follows: E. coli cells expressing water-soluble fluorescent protein are resuspended and homogenized in a lysis buffer containing 5mM imidazole, 20mM Tris-HCl pH 8.0 and 500mM NaCl. After centrifugation at 24000g, the supernatant is mixed with Ni 2+-NTA beads were incubated. After incubation, the beads were washed 3 times with washing buffer (20mM imidazole, 20mM Tris-HCl pH8.0 and 500mM NaCl), and then eluted with elution buffer (100mM imidazole, 20mM Tris-HCl pH 8.0 and 500mM NaCl). The eluted protein was then purified using a gel filtration column (Superdex200Increase 10 / 300GL, GE Healthcare) in TBS (pH 7.4). Parts of the target proteins wFAP0, wFAP1.1, wFAP1.2, and wFAP1.3 (amino acid sequences were SEQ ID No.: 6-9, respectively) were collected according to the A280 readings.

[0032] Example 2: Preparation and purification of transmembrane fluorescent protein

[0033] Transmembrane fluorescent proteins (tmFAPs) were constructed using pET-29b vector (with MBP (maltose binding protein) tag at the N-terminus and 6*His tag at the C-terminus). The plasmid was transferred into E. coli cells (Lemo21 (DE3) Competent E. coli, NEB) for expression and cultured at 37°C, 220RPM in LB medium. After OD reached 0.6, 0.2mM IPTG was added and cultured for 16 hours. For transmembrane fluorescent protein purification, cells were collected from at least 1L of E. coli culture expressing transmembrane fluorescent proteins, resuspended, and homogenized in 30ml lysis buffer (25mM Tris-HCl, pH 7.4, 150mM NaCl). Cells were lysed using a high-pressure homogenizer. Cell debris was removed by centrifugation at 15000g for 10 minutes. The supernatant was collected and ultracentrifuged at 25000g for 1 hour. The membrane fraction was solubilized with 1.5% n-Octyl-β-D-Glucopyranoside (OG, Anatracec) at 4°C for at least 2 hours, followed by centrifugation at 15,000 g for 45 minutes. The supernatant was loaded onto Ni 2+-NTA beads, washed with 20 ml washing buffer (lysis buffer plus 20 mM imidazole and 1.1% OG), and eluted with 10 ml elution buffer (washing buffer plus additional 300 mM imidazole). The eluted protein was concentrated to 1 ml and further purified by Superdex 200Increase 10 / 300GL gel filtration chromatography column (GE Healthcare). The gel filtration buffer contained 25 mM Tris-HCl, pH 7.4, 150 mM NaCl and 1.1% OG. According to the A280 readings and SDS-PAGE results, the target proteins tmFAP0.1, tmFAP1.1, tmFAP1.2, tmFAP1.3, and tmFAP2.1 (amino acid sequences were SEQ ID No.: 1-5, respectively) were collected.

[0034] Example 3: Determination of the fluorescence properties, affinity and specificity of the protein binding to HBC

[0035] Dissociation constant (Kd), fluorescence spectrum and extinction coefficient are measured using Varioskan LUX microplate detector (Thermo). All fluorescence readings are measured in black 96-well microplates (Corning). Protein concentration is estimated by measuring A280 using NanoPhotometer N60 (Implen). In order to determine Kd, a series of concentrations of purified protein (wFAP0, wFAP1.1, wFAP1.2, wFAP1.3, tmFAP0.1, tmFAP1.1, tmFAP1.2, tmFAP1.3) are mixed with 50nM HBC (HBC599, HBC620). The final total volume of each condition is 100 μL, containing 2% DMSO. Then measure the fluorescence reading at the optimal excitation wavelength. Use Origin Pro to fit the global dissociation constant. The fluorescence activation multiple of the protein is calculated using the data observed in the Kd titration experiment. The background fluorescence readings were compared to the steady-state fluorescence readings or the highest reading observed (for weak binders not measured at steady state). Fluorescence spectra of FAPs were measured using 2 μM HBCs and excess purified protein. 200 μL of sample was used for each protein-HBC combination. Fluorescence readings were scanned using the optimal excitation / emission wavelength for each HBC-protein combination. For tmFAP-HBC specific experiments, 1 μM protein was mixed with 1 μM HBC in each combination and the fluorescence reading was measured at the optimal wavelength for each condition.

[0036] Example 4: Extinction coefficient and absorption measurements

[0037] All absorbance readings were measured in transparent 96-well microplates using a Varioskan LUX microplate reader (Thermo). To measure the extinction coefficient, a series of concentrations of HBC derivatives were mixed with excess protein (the HBC derivative and protein combinations used were as follows: Figure 2 The mixture was incubated at room temperature for at least 3 hours and then added to a transparent 96-well microplate for absorption measurement. The absorption value at the absorption peak of each HBC-protein combination was used. These values ​​were processed by background subtraction of the absorption at 600nm, where the absorption of HBC is minimal, and path length correction was performed by measuring the absorption of water at 900nm and 975nm. The extinction coefficient was calculated using the Beer-Lambert-Bouguer law, and a linear fit was performed through the data points of at least 4 concentrations.

[0038] Example 5: Fluorescence quantum yield measurement

[0039] The absolute quantum yield was measured by using an integrating sphere on an Edinburgh FLS1000 spectrometer. 0.5 μM of HBC derivative was mixed with at least 2 μM of protein (the HBC derivative and protein combination used was Figure 2 The samples were mixed with 1% DMSO and incubated overnight at 4°C in the dark. All samples were measured at room temperature using an excitation wavelength of 495 nm.

[0040] The results of Examples 3-5 are shown in Figure 2-4 In, from Figure 2It can be seen that compared with EGFP (data from: Sarkisyan, K Set al. Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J 109, 380-389, doi: 10.1016 / j.bpj.2015.06.018 (2015).), the brightness and fluorescence quantum yield of the transmembrane fluorescent protein and water-soluble fluorescent protein of the present application are close to or exceed EGFP. The transmembrane fluorescent protein and water-soluble fluorescent protein of the present invention can specifically bind to HBC599 and HBC620 molecules after being expressed and purified in Escherichia coli. When the transmembrane fluorescent protein mutant binds to HBC599, the brightness is more than 1675 times that when no protein is added, the affinity reaches 17nM, the fluorescence quantum yield reaches 0.87, the shortest fluorescence emission peak is 555nm, and the longest is 590nm; when the water-soluble fluorescent protein mutant binds to HBC599, the brightness is more than 1150 times that when no protein is added, the affinity reaches 51nM, the fluorescence quantum yield reaches 0.88, the shortest fluorescence emission peak is 545nm, and the longest is 590nm.

[0041] Example 6: Crystalline Structure

[0042] crystallization

[0043] For protein crystallization, purified wFAP1.1 was concentrated to 12 mg / ml. Crystals were obtained by hanging drop evaporation diffusion at 18°C. To prepare hanging drops, 0.8 μL of concentrated protein solution was mixed with 0.8 μL of crystallization solution containing 20% ​​PEG1000, 100 mM disodium phosphate / citric acid pH 4.2 and 200 mM lithium sulfate. For crystallization of the wFAP1.1 / HBC599 complex, wFAP1.1 was mixed with 2% volume / volume of 50 mM HBC599. Crystals were obtained under the same conditions as the single protein X-ray diffractometer.

[0044] Data acquisition and structure determination

[0045] Prior to X-ray diffraction experiments, crystals were soaked in crystallization solution containing additional 20% vol / vol glycerol for protection. Diffraction data for wFAP1.1 apo were collected on a single crystal X-ray diffractometer at the Biomedical Platform of Westlake University. Diffraction data for wFAP1.1 / HBC599 complex were collected at the BL02U1 beamline of the Shanghai Light Source. Data were processed and scaled using HKL2000 software.

[0046] The wFAP1.1 apo and wFAP1.1 / HBC599 structures were determined by molecular replacement in PHASER using the designed model as the search model. The structural models were refined in PHENIX and adjusted in COOT. The model quality was checked by MolProbity. All structural figures were made using PyMOL (https: / / www.pymol.org).

[0047] Figure 5 The crystal structures of the water-soluble fluorescent protein wFAP1.1 in the state of binding to HBC and in the state of not binding to HBC are shown. It can be seen from the figure that the fluorescent group HBC599 is tightly bound to the protein in the core part of the protein.

[0048] Example 7: Human cell culture, transfection and fluorescence imaging

[0049] For human cell imaging, all protein constructs used pcDNA vectors (with Ig Kappa tags and neurexin signal peptides at the N-terminus of the protein and mtagBFP2 at the C-terminus for display of protein localization in cells). HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, ThermoFisher) supplemented with 10% fetal bovine serum (Cellmax) and 50U / ml of penicillin and streptomycin. The constructed plasmid vectors were transfected into the cells using Lipofectamine 3000 transfection reagent (ThermoFisher) according to the manufacturer's instructions. One day after transfection, the cells were washed once with PBS buffer and then replaced with PBS buffer containing 1% HBC599. Subsequently, the cells were incubated for 10 minutes and imaged using a Nikon C2 confocal microscope.

[0050] Example 8: Live Cell Imaging in E. coli

[0051] E. coli cells expressing tmFAP or wFAP were collected by centrifugation from the culture medium of Examples 1 and 2, washed twice with M9 medium, and incubated with 200 nM HBC599 (1% DMSO) for at least 20 minutes before imaging. Fluorescence imaging was performed using the Airyscan mode of a Zeiss 980 microscope, using a 63x oil lens. 405 nm and 488 nm lasers were used to excite mtagBFP2 and HBC599 signals, respectively.

[0052] Figure 6 Example 7 ( Figure 6 b) and 8( Figure 6a), it can be seen from the figure that the fluorescent signal is localized on the cell membrane and has a high signal-to-noise ratio, while cells without protein expression (negative control) have no fluorescent signal under the same conditions. These results show that the protein of the present application can be used for fluorescence imaging in cells and on the cell membrane.

[0053] Example 9: Detection of the effects of wFAP1.1 and tmFAP1.1 mutations on fluorescence activation

[0054] For single point mutations, mutations were introduced into the corresponding sites by PCR. The mutants were then transformed into Escherichia coli BW28873 cells by electroporation and cultured in SOB medium (Sigma) at 37°C. Both wFAP1.1 and tmFAP1.1 were induced by 0.0004% arabinose for 3.5 hours. The cells were then washed three times with TBS buffer and then incubated with HBC599 (1% DMSO) at room temperature for more than 10 minutes. Flow cytometry (Melody, BD) was used to sort cells according to the fluorescence signals of HBC599 and mtagBFP2. All samples were incubated with 200nM HBC599 for the same time. 3000 events were recorded for each mutant for analysis.

[0055] The results are as follows Figure 7 As shown. Figure 7 It can be seen that the mutations introduced into the HBC binding pocket of wFAP1.1 and tmFAP1.1 have significantly reduced fluorescence compared to the unmutated protein. This result can indicate that the protein of the present application produces highly specific, high-affinity binding with the HBC molecule through the amino acids in the core region, rather than non-specific weak binding.

Claims

1. A transmembrane fluorescent protein, wherein the amino acid sequence of the transmembrane fluorescent protein is SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4 or SEQ ID No.:

5.

2. A water-soluble fluorescent protein corresponding to the transmembrane fluorescent protein according to claim 1, wherein the amino acid sequence of the water-soluble fluorescent protein is SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.:

9.

3. A polynucleotide encoding the transmembrane fluorescent protein according to claim 1 or the water-soluble fluorescent protein according to claim 2. An expression vector comprising the polynucleotide according to claim 3 . A cell comprising the expression vector according to claim 4 . The cell according to claim 5 , which comprises an E. coli cell or a human HEK293 cell.

7. Application of the transmembrane fluorescent protein as claimed in claim 1 in imaging and sensing on cell membranes.

8. Use of the water-soluble fluorescent protein as claimed in claim 2 in cell imaging and sensing.

9. A method for cell membrane imaging, comprising using the transmembrane fluorescent protein as claimed in claim 1.

10. A cell imaging method, comprising using the water-soluble fluorescent protein according to claim 2.

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