BDFPs mutant proteins, fusion proteins and applications

Abstract

The present invention discloses BDFPs mutant proteins, fusion proteins and applications. The present invention provides a variety of small molecule BDFPs mutant proteins, wherein a mutant protein with an emission wavelength of about 663nm to 704nm and high brightness is obtained by site mutation. The amino acid sequence of the BDFPs mutant protein is shown in SEQ ID NO.5 to SEQ ID NO.23. The BDFPs mutant proteins obtained by site mutation in the present invention are mostly monomeric structures, have a smaller molecular weight, and their effective brightness is improved. The present invention provides more choices for fluorescent proteins for deep imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent markers, and in particular relates to BDFPs mutant proteins, fusion proteins and applications. Background Art

[0002] In recent years, with the rapid development of near-infrared fluorescence imaging technology and its successful application in the biological industry, wide-field fluorescence microscopes and confocal fluorescence microscopes developed based on the principles of charge coupled device (CCD) imaging systems and laser imaging systems have been successfully applied to in vivo imaging of cell tissues. This technology has become an important research method for studying neural tissue cells, cell transduction processes and protein markers, especially in the clinical diagnosis of tumor cells.

[0003] Fluorescent proteins work best with light at 650-900nm when performing deep tissue imaging and in vivo imaging. The light penetration in this wavelength range is stronger than that in the blue light region, and the autofluorescence of light in this range can be basically ignored, and the light reflection ability is also very low (Weissleder and Biotechnology 2001). Traditional fluorescent proteins have a shorter fluorescence wavelength and weaker cell penetration ability, and animal cells have strong background fluorescence. Labeling living animal tissue cells cannot capture their images well, so it is more urgent to evolve red or near-infrared fluorescent proteins with longer wavelengths that can reduce light loss and improve the sensitivity of in vivo imaging. In addition, the existing fluorescent proteins that can excite far-red or near-infrared light have a large molecular weight, are prone to aggregation and precipitation during in vivo detection, and are not tolerant to extreme environments, so their application occasions are limited.

[0004] Recently, we reported the generation and properties of far-red (FR) and near-infrared (NIR) fluorescent phycobiliproteins, termed BDFPs. BDFPs do not covalently bind phycocyanobilin (PCB) as their progenitor, the f-subunit of allophycocyanin (APCF), but rather covalently bind the more accessible biliverdin (BV). Far-red BDFPs fluoresce most strongly at ∼670 nm, and near-infrared BDFPs fluoresce most strongly at ∼710 nm.

[0005] However, making fluorescent proteins (FPs) smaller and improving their effective brightness remains a challenge. Summary of the invention

[0006] The first object of the present invention is to provide a BDFPs mutant protein with better protein molecular weight and better effective brightness.

[0007] The second object of the present invention is to provide a fusion fluorescent protein.

[0008] The third object of the present invention lies in the relevant biological materials.

[0009] The fourth object of the present invention is to provide applications of the above-mentioned BDFPs mutant protein or fusion protein or related biomaterials.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned BDFPs mutant protein or fusion protein or related biological materials in the preparation of products.

[0011] A sixth object of the present invention is to provide a product.

[0012] The seventh object of the present invention is to provide a method for locating the expression of a target protein in a cell or a living animal.

[0013] The technical solution adopted by the present invention is:

[0014] A BDFPs mutant protein, wherein the BDFPs mutant protein is any one of the following:

[0015] (a1) The BDFPs mutant protein comprises a mutation in at least one of positions 23, 24, 27, 30, 31, 38, 58, 68, 81, 83, 85, 87, 88, 91, 92, 107, 112, 116, 119, 127, and 130 of the BDFP fluorescent protein and / or a deletion of one or more amino acids selected from positions 20-31, wherein the sequence of the BDFP fluorescent protein is shown in SEQ ID NO.2;

[0016] (a2) the BDFPs mutant protein comprises a mutation in at least one of positions 23, 48, 49, 53, 58, 68, 70, 77, 87, 108, 127, 144, 149 of the BDFP fluorescent protein and / or a deletion of one or more amino acids selected from positions 20-31, wherein the sequence of the BDFP fluorescent protein is shown in SEQ ID NO.3;

[0017] (a3) The BDFPs mutant protein includes at least one mutation at positions 58 and 68 of the BDFP near-infrared fluorescent protein, wherein the sequence of the BDFP fluorescent protein is shown in SEQ ID NO.4.

[0018] In some embodiments of the present invention, the valine (V) at position 23 is mutated to lysine (K), the valine (V) at position 24 is mutated to arginine (R), the leucine (L) at position 27 is mutated to aspartic acid (D) or glutamine (Q), the leucine (L) at position 30 is mutated to histidine (H) or glutamine (Q), the leucine (L) at position 31 is mutated to glutamic acid (E) or glutamine (Q), the valine (V) at position 38 is mutated to serine (S) or arginine (R), the alanine (A) at position 48 is mutated to glutamine (Q), and the valine (V) at position 49 is mutated to arginine (R). The 49th alanine (A) mutated to valine (V), the 53rd lysine (K) mutated to glutamine (Q), alanine (A), glycine (G) or arginine (R), the 58th leucine (L) mutated to glutamine (Q), the 68th serine (S) mutated to arginine (R), the 70th glycine (G) mutated to aspartic acid (D), the 77th arginine (R) mutated to histidine (H), the 81st methionine (M) mutated to alanine (A), isothiocyanate (A), thiazolyl (D), ...thiazolyl (D), arginine (R) mutated to histidine (H), the 58th leucine (L) mutated to glutamine (Q), the 68th serine (S) mutated to arginine (R), the 70th glycine (G) mutated to aspartic acid (D), the 77th arginine (R) mutated to histidine (H), the 81st methionine (M) mutated to alanine (A), isothiocyanate (A), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), thiazolyl (D), Leucine (I), threonine (T) or lysine (K), valine (V) at position 83 mutated to histidine (H), aspartic acid (D) at position 85 mutated to glycine (G), asparagine (N) at position 87 mutated to serine (S) or aspartic acid (D), tyrosine (Y) at position 88 mutated to arginine (R), arginine (R) at position 91 mutated to histidine (H), methionine (M) at position 92 mutated to lysine (K), glycine (G) at position 107 mutated to glutamic acid (E), Arginine (R) at position 8 mutated to glutamine (Q), glycine (G) at position 112 mutated to lysine (K), threonine (T) at position 116 mutated to alanine (A), serine (S) at position 119 mutated to glycine (G), alanine (A) at position 127 mutated to valine (V), glycine (G) at position 130 mutated to alanine (A), threonine (T) at position 144 mutated to alanine (A) or arginine (R), and asparagine (N) at position 149 mutated to aspartic acid (D).

[0019] In some preferred embodiments of the present invention, one or more amino acids are deleted from positions 20-24.

[0020] In some preferred embodiments of the present invention, the amino acid sequence of the BDFPs mutant protein is shown as SEQ ID NO.5 to SEQ ID NO.23.

[0021] The second aspect of the present invention provides a fusion protein, comprising the BDFPs mutant protein described in the first aspect of the present invention.

[0022] The third aspect of the present invention provides a biological material related to the mutant protein described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention, wherein the biological material is any one of the following (b1) to (b8):

[0023] (b1) a nucleic acid molecule encoding the mutant protein or the fusion protein;

[0024] (b2) an expression cassette containing the nucleic acid molecule described in (b1);

[0025] (b3) a recombinant vector containing the nucleic acid molecule described in (b1);

[0026] (b4) a recombinant vector containing the expression cassette described in (b2);

[0027] (b5) a recombinant microorganism containing the nucleic acid molecule described in (b1);

[0028] (b6) a recombinant microorganism containing the expression cassette described in (b2);

[0029] (b7) a recombinant microorganism containing the recombinant vector described in (b3);

[0030] (b8) A recombinant microorganism containing the recombinant vector described in (b4).

[0031] In some embodiments of the present invention, the recombinant microorganism is a eukaryotic or prokaryotic microorganism well known in the art such as Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae or Bacillus subtilis that can be used to express the target protein.

[0032] The fourth aspect of the present invention provides the use of the mutant protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, or the related biological material described in the third aspect of the present invention in any one of (c1) to (c7):

[0033] (c1) In vivo imaging;

[0034] (c2) Cell localization;

[0035] (c3) acting as a fluorescence resonance energy transfer acceptor or donor;

[0036] (c4) Optical imaging studies;

[0037] (c5) gene expression;

[0038] (c6) preparing fluorescent probes;

[0039] (c7) Cell screening.

[0040] The fifth aspect of the present invention provides the use of the mutant protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, or the related biomaterial described in the third aspect of the present invention in the preparation of a product, wherein the function of the product is any one of (c1) to (c7):

[0041] (c1) In vivo imaging;

[0042] (c2) Cell localization;

[0043] (c3) acting as a fluorescence resonance energy transfer acceptor or donor;

[0044] (c4) Optical imaging studies;

[0045] (c5) gene expression;

[0046] (c6) preparing fluorescent probes;

[0047] (c7) Cell screening.

[0048] The sixth aspect of the present invention provides a product comprising the BDFPs mutant protein described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0049] In some embodiments of the invention, the product is a kit.

[0050] The seventh aspect of the present invention provides a method for locating the expression of a target protein in a cell or a living animal, by fusing the target protein gene with the nucleic acid molecule described in the third aspect of the present invention and inserting the resultant into a suitable expression vector; then transfecting the expression vector into a cell or a living body, and observing the expression location of the target protein in the cell or living animal under an excitation spectrum.

[0051] The beneficial effects of the present invention are:

[0052] The present invention provides a variety of small molecule BDFPs mutant proteins, wherein a mutant protein with an emission wavelength of about 663nm to 704nm and high brightness is obtained by site mutation. The maximum fluorescence emission peak of the existing BDFP near-infrared fluorescent protein is near 710nm (near-infrared light). The present invention obtains a blue shift in the fluorescence emission peak of the BDFP obtained by site mutation, wherein the maximum fluorescence emission wavelength of a part of the protein is basically about 670nm (far-infrared light); the other part, such as BDFP2.1, BDFP2.2, BDFP2.5, and BDFP2.6, is about 700nm, which is a near-infrared fluorescent protein. Moreover, the obtained BDFPs mutant proteins are mostly monomeric structures, such as BDFP2.1, BDFP2.2, BDFP2.5, BDFP2.6, B, D, E, M, N, O, and U, with a smaller molecular weight, and its effective brightness is improved. The present invention provides more choices for fluorescent proteins for deep imaging, and the fluorescent protein provided by the present invention can be used in combination with other fluorescent proteins, and small molecule fluorescent proteins are more suitable as protein fusion tag sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 For homologous structure simulation analysis. Figure 1 a is the structural simulation analysis of mutant V2, yellow represents the amino acid sites related to the covalent binding of biliverdin BV, magenta represents the amino acid sites around cysteine ​​Cys72 and / or Cys82 facing the surface of the pigment group, blue represents the amino acid sites not facing the surface of the pigment group, and orange represents the ring structure (SWISS-MODEL, template using ApcB (PDB:1ALL)); Figure 1 b is the structural simulation analysis of BDFP2.1, and bright pink represents the amino acid sites related to the effective brightness improvement (SWISS-MODEL, template using ApcB (PDB:1ALL)) Figure 1 c is the simulation analysis of the dimer structure of BDFP1.6 (SWISS-MODEL, using ApcB (PDB:1ALL) as the template), where labels A and B represent different peptide chains; Figure 1 d is the homologous structure comparison of mutant V2 (gray) and smURFP(Y56R) (PDB:6FZN) (wheat color).

[0054] Figure 2 To express BDFPs fusion protein in living Hela cells under STED nanoscopy. Figure 2 a is Tomm20:BDFP2.5; Figure 2 b is Keratin:BDFP2.6; Figure 2 c is MTS:BDFP2.6; Figure 2d is STED dual-color microscopy, using far-red and near-infrared light BDFPs to mark the location of human proteins in cells. Scale bar 10 μm.

[0055] Figure 3 Analyze the aggregation state of molecular sieve layers. Figure 3 a is sample (BDFP2.1-2.6); Figure 3 b is the standard sample.

[0056] Figure 4 For expression of bdfps:IRES:eGFP fusion protein in HEK 293T cells under the same conditions, the expression level was first corrected using the mean fluorescence intensity of eGFP and then referenced to the mean fluorescence intensity of BDFP1.6. Error bars, SEM (number of images n=5).

[0057] Figure 5 To express the bdfps:IRES:eGFP fusion protein in HEK 293T cells under the same conditions, the expression level was first corrected using the mean fluorescence intensity of eGFP and then compared to the mean fluorescence intensity of miRFP670nano. Error bars, SEM (number of images n = 10).

[0058] Figure 6 Effective brightness contrast of mutant AX. DETAILED DESCRIPTION

[0059] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0060] Experimental methods

[0061] 1. Cloning and plasmid construction

[0062] For specific operations, please refer to the standard experimental manual "Molecular Cloning", second edition, published by Cold Spring Harbor Laboratory in 1989.

[0063] pET28 and pACYCDuet (Novagen) are T7 promoter expression vectors. pACYCDuet is designed to be used for co-transformation and expression of two target gene sequences in E. coli.

[0064] The gene encoding the fluorescent protein sequence can be cloned into the pET28a vector through the restriction sites NcoI and XhoI. The heme oxygenase gene ho1 can be cloned into the pACYCDuet vector plasmid for the production of biliverdin BV.

[0065] The expression vector pcDNA3.1 (Invitrogen) is a mammalian expression vector with a CMV promoter.

[0066] When screening in HEK 293T cells, the expression vector pcDNA3.1 was used, and the fusion expression sequence was designed to be FP:IRES:eGFP. When making brightness comparisons, the fluorescence brightness of the FP protein can be corrected based on the fluorescence brightness of eGFP.

[0067] When super-resolution microscopy was performed on the smooth endoplasmic reticulum, the cytochrome P450 transmembrane domain gene and the N-terminal linker gene were cloned into pcDNA3.1 to obtain pcDNA3.1-CytERM-linker, and bdfps was inserted downstream of CytERM.

[0068] 2. E. coli Expression

[0069] After the pET expression vector encoding the fluorescent protein was transformed into the E. coli strain BL21 (DE3) (Novagen), the vector pACYC-ho1 was transformed into the same strain. The transformed BL21 cells were cultured at 18°C ​​in LB medium supplemented with kanamycin (20 μg / ml) and chloramphenicol (17 μg / ml). When the OD value reached 0.4-0.6, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was used to induce expression for 5-16 hours, and then centrifuged at 4°C, 12,000×g for 3 minutes, the cells were collected, washed twice with water, and stored at 4°C for a short period of time or at -20°C for a long period of time.

[0070] 3. Mammalian Cell Transfection and Observation

[0071] HEK 293T or HeLa cells were cultured in DMEM medium (Invitrogen) containing 10% fetal bovine serum. 3000 (Invitrogen) for transfection. 2000 and DNA at a ratio of 2:1 (μl:μg) in serum-free medium After mixing for 10 minutes, add it to the cells to be transfected. After 6 to 8 hours, replace with fresh DMEM medium.

[0072] Live cell imaging (microscopy) was performed 24 hours after the fluorescent protein was expressed. Before imaging, the cells were rinsed twice with 1 mL PBS and then once with DEME medium (without phenol red). The imaging equipment was an inverted microscope Nikon Ti equipped with a cool-snap HQ2 CCD camera and a Nikon Plan Fluor ELWD 20 0.45-DIC L-WD objective.

[0073] The excitation and emission settings were as follows: eGFP was the green channel, ex = 470 / 40, em = 510 / 40 nm; the far-red light BDFPs protein was the far-red light channel, ex = 630 / 20, em = 690 / 50 nm; the near-infrared light BDFPs protein and IFP2.0 were the near-infrared light channel 1, ex = 650 / 40, em = 710 / 50 nm; and the iRFP720 was the near-infrared light channel 2, ex = 650 / 40, em = 720 / 40 nm.

[0074] Images were analyzed and processed using ImageJ software (National Institutes of Health).

[0075] 4. Protein Purification and Quantification

[0076] The wet cells were suspended in ice-cold starting buffer [potassium phosphate buffer (KPB, 20 mM, pH 7.2), sodium chloride (NaCl, 0.5 M)]. The cells were disrupted by 50 W ultrasound (JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd., China) for 5 minutes. The suspension was centrifuged at 4 °C and 12,000 × g for 60 minutes. The supernatant was purified by Ni 2+ The protein was purified by affinity chromatography column (Amersham Biosciences) using starting buffer [potassium phosphate (KPB, 20 mM, pH 7.2) for loading and elution with buffer containing 0.5 M imidazole. The collected samples were dialyzed at least twice with starting buffer (pH 7.2). The protein concentration was determined by Bradford method and calibrated using bovine serum albumin as a standard.

[0077] 5. Homology Analysis

[0078] The BDFP fluorescent protein structure alignment was completed on the SWISS-MODEL remote server. The comparison software was Swiss-PDB Viewer. PyMOL (http: / / www.pymol.org / ) was used to create the protein structure diagram. Clustal (http: / / www.clustal.org / ) was used to create the protein sequence alignment diagram.

[0079] 6. Protein oligomerization status analysis

[0080] The molecular weight of the protein sample can be determined by molecular sieve purification and compared with a group of protein markers (12–66 kDa; Sigma–Aldrich), and its oligomeric state can be inferred. The protein sample loading volume is 1 mL, and the sample is purified by Ni2+ affinity chromatography and dialyzed into KPB buffer (20 mM, pH 7.2, containing 150 mM NaCl). The molecular sieve column type is Superdex 75 (30×1.0 cm), and the elution buffer conditions are the same as the sample.

[0081] 7. Spectral analysis

[0082] The absorption spectrum of fluorescent proteins was detected using a DU-800 spectrophotometer produced by Beckman-Coulter.

[0083] The extinction coefficient of fluorescent protein is based on the absorption coefficient of bile pigment BV at 390nm ε = 39,900M -1 cm -1 , for reference conversion.

[0084] The fluorescence spectrum was detected by a fluorescence spectrophotometer (F320, Tianjin Gangdong Technology Development Co., Ltd.), and the sample detection environment was potassium phosphate solution (20mM, pH 7.2, KPB). F The calculation was based on the respective maximum fluorescence, with iRFP670 (F = 0.122) as the reference value. When calculating the molecular brightness, refer to iRFP670 nano, isε·Φ fl =10.3mM -1 cm -1 .

[0085] 8. Super-resolution microscopy imaging

[0086] Wide-field and structured illumination microscopy (SIM) images were acquired at room temperature using a Nikon structured illumination system on an ECLIPSE Ti-E inverted Nikon microscope equipped with a 100×1.49NA oil immersion objective in standalone mode. NIR fluorescence was excited using a 640nm semiconductor laser (100mW, CUBE 640-100C, COHERENT). Data were acquired using an electron multiplying CCD camera (Andor iXon3 DU897) controlled by NIS-Elements AR software (Nikon). Images were processed using NIS-ElementsAR.

[0087] The experiment of observing BDFPs fusion protein at room temperature was carried out using a Leica STED 775nm scanning microscope (Leica TCS SP8 3X, Leica Microsystems, Wetzlar, DE), which was equipped with a HCPL APO 100×1.4NA oil immersion objective lens set and a gated HyD detector.

[0088] The BDFPs fusion protein was first excited with a 40MHz 640nm pulsed diode laser, and then depleted with a 40MHz 775nm pulsed laser. The depletion laser beam was constrained into a donut shape in the focal plane by a spatial light modulator (LCOS-SLM X10468, Hamamatsu Photonics). The excitation and depletion laser beams were coupled together and then scanned with a fast galvanometer mirror. After the fluorescence signal was decoupled, it was focused onto a photon counter (SPCM-AQRH-13, Excelitas Technologies) through a confocal pinhole (about 1 Airy disk unit).

[0089] The raw data images will be processed by spectral decomposition and deconvolution using Huygens professional (Scientific Volume Imaging). Image processing will be performed using Leica Application Suite X (LAS X) software.

[0090] 9. Quantitative and statistical analysis

[0091] All fluorescence photos were analyzed using ImageJ (National Institutes of Health) software, and data graphs and statistics were generated using Origin 8.0 (OriginLab) software.

[0092] Example 1

[0093] 1. BDFPs were constructed using BDFP1.6 as a template. The construction process of BDFP1.6 protein was based on the Chinese patent application "Genetically modified novel BDFP fluorescent protein and its fusion protein" (application number 2018110097266), and its sequence was: ApcF2 (20-169) -F30L / S46T / I51V / N72C / Y82C / Y92M / D101G / E107G / L109M / L113F / G125C / T127A / S130G / N136K / V143A / T151A / V160I / V161A / E163V. BDFP1.6 protein is BDFP far-red fluorescent protein.

[0094] Unless otherwise specified, the amino acid positions in the present invention are encoded based on the protein sequence of the phycobiliprotein ApcF2. The amino acid sequence of the phycobiliprotein ApcF2 of the present invention is shown in SEQ ID NO.1.

[0095] (1) Using BDFP1.6 as a template, amino acids 20-31 of BDFP1.6 were deleted to obtain V1, i.e., BDFP1.6 (32-169) protein, to achieve monomerization ( Figure 3 );

[0096] (2) Then, using V1 as a template, several rounds of random mutagenesis were performed by error-prone PCR. The better mutation sites screened out in each round of mutagenesis were summarized, and then L58Q and S68R mutations were performed to obtain V2.

[0097] (3) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0098] (4) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0099] (5) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0100] The results showed that the effective brightness of V2 with L58Q and S68R mutations was increased by 2.2 times compared with V1 ( Figure 4 ).

[0101] The sequence table of all BDFPs mutants involved in the examples of this application is shown in Table 1.

[0102] Table 1 Sequence list of BDFPs mutants

[0103]

[0104]

[0105]

[0106] The PCR primers used for gene construction are shown in Table 2.

[0107] Table 2 Some PCR primers used for gene construction

[0108]

[0109]

[0110]

[0111]

[0112] The comparison of the basic properties of BDFPs mutants is shown in Table 3.

[0113] Table 3 Comparison of basic properties of different mutants

[0114]

[0115] The basic properties of different mutants are shown in Table 4.

[0116] Table 4 Comparison of basic properties of BDFPs mutants

[0117]

[0118]

[0119] Example 2

[0120] (1) The three-dimensional structure of the mutant protein was simulated and the amino acid homologous sequences (BDFP1.6, smURFP, ApcB, etc.) were compared to find the key amino acid mutation sites ( Figure 1 ).

[0121] Among them, M81, D85, Y88, M92 and T116 are facing the surface of the pigment group and are quite close to the pigment (about ).

[0122] G57 in smURFP corresponds to amino acid N87 in BDFPs ( Figure 1 d).

[0123] In addition, the BDFPs mutation G112K introduces positive charge, and the mutation A127V enhances the affinity between the pigment group and the protein through van der Waals interaction.

[0124] (2) Directed mutagenesis or partial saturation mutagenesis: Mutation to positively charged amino acids (H, R, K) resulted in mutants M81K, V83H, Y88R, M92K, and partial saturation mutagenesis of M81 to amino acids (A, I, T) resulted in V4-V11.

[0125] Then, based on mutant V7 (M81K), further site-directed mutations were performed on D85, N87, R91, G112, S119, and A127V to obtain V13-V18.

[0126] (3) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0127] (4) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0128] (5) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0129] Among them, M81K (v7) has the best effective brightness, which is 2.4 times higher than v1, and the spectrum has a 5nm blue shift ( Figure 4 ).

[0130] The next best choices are V14 (N87), V16 (G112K), and V18 (A127V).

[0131] The effective brightness of mutants v4-v6 (M81A, M81I and M81T) dropped sharply or even disappeared.

[0132] Example 3

[0133] (1) Using V1 as a template, L58Q, S68R, and M81K point mutations were introduced to obtain BDFP2.1 (V19).

[0134] (2) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0135] (3) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0136] (4) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0137] The effective brightness in HEK 293T cells was 3.5 times higher than that of v1 and 1.36 times higher than that of miRFP670nano (Table 3). In addition, BDFP2.1 (V19) is a monomer with a molecular weight of 15 kDa, which is slightly smaller than miRFP670nano (17 kDa). Figure 5 )

[0138] Example 4

[0139] (1) Using BDFP1.6 as a template, we first mutated the hydrophilic amino acids L27 and L31 in BDFP1.6 to generate a new variant v20 (Table 3, Figure 5 ). Gel filtration through a Superdex 75 column showed a mixture of monomers and dimers.

[0140] We further mutated all hydrophobic amino acids on the N-terminal helix, including V23, V24, L27, L30, L31, and V38 in BDFP1.6 ( Figure 1 c), and obtained monomeric proteins (V21-V22), which were verified by molecular sieve gel filtration chromatography experiments.

[0141] Obviously, mutating the hydrophobic amino acids in the dimer binding region into hydrophilic amino acids will promote protein monomerization ( Figure 3 ).

[0142] Finally, based on V21, V23K, L58Q and S68R mutation sites were supplemented, and the depolymerization effects of V23K / V24R / L27Q / L30Q / L31Q / V38R and the effective brightness improvement effect of L58Q / S68R / M81K were integrated to obtain BDFP2.2 (V23).

[0143] (2) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0144] (3) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0145] (4) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0146] The effective brightness of BDFP2.2 (V23) in HEK293T cells is 2.1 times that of BDFP2.1 (V19) and 2.8 times that of miRFP670nano. Comparison of BDFP2.2 and BDFP2.1 shows that the N-terminal has a higher brightness ( Figure 5 ).

[0147] Example 5

[0148] (1) Using BDFP2.2 as a template, amino acids 20-24 of BDFP were deleted to obtain BDFP2.3 (V24).

[0149] (2) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0150] (3) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0151] (4) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0152] The effective brightness of BDFP2.3 in HEK293T cells was 1.5 times that of BDFP2.1 and it had higher photostability than BDFP2.1 and BDFP2.2. In addition, BDFP2.3 produced little precipitation after being dialyzed in KPB buffer (20 mM, pH 7.2, containing 0.15 M NaCl) for 12 h.

[0153] The effective brightness of BDFP2.1, 2.2 and 2.3 in HEK 293T cells was 1.4, 2.9 and 2 times that of miRFP670nano, respectively ( Figure 5 , Table 3).

[0154] Example 6

[0155] In this embodiment, BDFP1.9 is used as a template, wherein the construction process of BDFP1.9 protein refers to the Chinese patent application "A small molecule near-infrared fluorescent protein and its fusion protein" (application number 2019105868013), wherein BDFP1.9 is also mutated from BDFP1.6, equivalent to BDFP1.6 (V24R / L27Q / L30Q / L31Q / V38R / M81K / F113L / C125G / A127V). BDFP1.9 protein is BDFP near-infrared fluorescent protein.

[0156] (1) Using monomeric BDFP1.9 (20-169) fluorescent protein as a template, mutation was performed by error-prone PCR, random mutagenesis kit, etc. to obtain BX. The specific mutant sequence is shown in Table 1, where A is BDFP1.9.

[0157] (2) The mutated sequence is used to construct a prokaryotic expression system, where it is expressed and covalently bound to the BV pigment.

[0158] (3) Protein purification and protein data analysis will then be carried out, and its absorption and fluorescence spectra will be measured, and relevant parameters will be calculated.

[0159] (4) The constructed mutants are screened in large quantities by transfecting animal cells to select mutants with higher effective brightness.

[0160] The results showed that the single mutation sites such as K53Q, K53A, K53G, K53R, and T144A increased by 1.2-1.5 times, and the effective brightness of mutants K53A, K53G, K53A / T144A, and K53G / T144A increased by 1.43, 1.32, 1.5, and 1.57 times, respectively, compared with BDFP1.9. Moreover, size exclusion chromatography confirmed that these mutants were monomers (Table 4, Figure 6 ).

[0161] Example 7

[0162] (1) BDFP1.9 was completely deleted for the N-terminal helix (aa20-31), followed by mutations L58Q and S68R to generate BDFP2.4 (V25), the effective brightness of which was 1.1 times that of BDFP1.9 ( Figure 5 , Table 3).

[0163] (2) BDFP1.9 was directly mutated at positions 23, 58, and 68 (V23K, L58Q, and S68R) to generate BDFP2.5 (V26), which is 1.5 times brighter than BDFP1.9 (Table 3).

[0164] (3) In BDFP2.5, aa20-25 on the N-terminal helix were deleted, resulting in a new mutant BDFP2.6 (V27), whose effective brightness is 1.4 times that of BDFP1.9 ( Figure 5 , Table 3).

[0165] Example 8

[0166] L58Q, S68R and M81K were introduced into BDFP1.6 and BDFP1.8, wherein the construction process of BDFP1.8 protein refers to the Chinese patent application "A near-infrared fluorescent protein and its fusion protein" (application number 2019105868028). BDFP1.8 is also mutated from BDFP1.6, which is equivalent to BDFP1.6 (M81K / F113L / C125G / A127V). BDFP1.8 protein is BDFP near-infrared fluorescent protein.

[0167] BDFP2.7 (V28) and BDFP2.8 (V29) were generated respectively. The effective brightness of these two new BDFPs is increased by 1.3 times ( Figure 5 , Table 3).

[0168] Example 9

[0169] In recent years, monomeric near-infrared fluorescent proteins derived from bacterial phytochromes have performed well in nanoscale STED imaging. In order to evaluate the excellent photostability and high brightness of BDFPs, STED imaging of BDFPs is an effective demonstration method.

[0170] The applicant selected these monomeric BDFPs with better photostability and brightness, and conducted STED imaging experiments using HeLa living cells. The fusion expression proteins were Keratin:BDFP2.6, Tomm20:BDFP2.5, and MTS:BDFP2.6 ( Figure 4 ).

[0171] BDFP2.5 and BDFP2.6 monomers have fluorescence peaks at 694nm and 703nm, respectively, and can be used as fusion protein markers for STED imaging under 640nm excitation and 775nm depletion wavelengths.

[0172] In addition, BDFP2.3:H2B and Keratin:BDFP1.9 can be easily adjusted to achieve dual-color STED imaging ( Figure 2 ).

[0173] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A BDFPs mutant protein, the amino acid sequence of the BDFPs mutant protein is shown in any one of SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.

19.

2. A fusion protein comprising the mutant protein according to claim 1.

3. A biological material related to the mutant protein according to claim 1 or the fusion protein according to claim 2, wherein the biological material is any one of the following (b1) to (b7): (b1) a nucleic acid molecule encoding the mutant protein or the fusion protein; (b2) an expression cassette containing a nucleic acid molecule encoding the mutant protein or the fusion protein; (b3) a recombinant vector containing a nucleic acid molecule encoding the mutant protein or the fusion protein; (b4) a recombinant vector containing an expression cassette, wherein the expression cassette is an expression cassette containing a nucleic acid molecule encoding the mutant protein or the fusion protein; (b5) a recombinant microorganism containing a nucleic acid molecule encoding the mutant protein or the fusion protein; (b6) a recombinant microorganism containing an expression cassette, wherein the expression cassette is an expression cassette containing a nucleic acid molecule encoding the mutant protein or the fusion protein; (b7) A recombinant microorganism containing a recombinant vector, wherein the recombinant vector is a recombinant vector containing a nucleic acid molecule encoding the mutant protein or the fusion protein.

4. Use of the mutant protein according to claim 1, the fusion protein according to claim 2, or the related biological material according to claim 3 in any one of the following (c1) to (c6): (c1) In vivo imaging; (c2) Cell localization; (c3) as fluorescence resonance energy transfer acceptor or donor; (c4) gene expression; (c5) preparing fluorescent probes; (c6) Cell screening; The application is for non-diagnostic treatment purposes.

5. The use according to claim 4, characterized in that: The in vivo imaging includes optical imaging studies.

6. Use of the mutant protein according to claim 1, the fusion protein according to claim 2, or the related biomaterial according to claim 3 in the preparation of a product, wherein the function of the product is any one of (c1) to (c6): (c1) In vivo imaging; (c2) Cell localization; (c3) as fluorescence resonance energy transfer acceptor or donor; (c4) gene expression; (c5) preparing fluorescent probes; (c6) Cell screening.

7. The use according to claim 6, characterized in that: The in vivo imaging includes optical imaging studies.

8. A product comprising the mutant protein according to claim 1 or the fusion protein according to claim 2 or the related biological material according to claim 3.

9. A method for locating the expression of a target protein in a cell or a living animal, characterized in that: The target protein gene and the nucleic acid molecule encoding the mutant protein or the fusion protein as described in claim 3 are fused and inserted into a suitable expression vector; the expression vector is then transfected into cells or living bodies, and the expression location of the target protein in cells or living animals is observed under the excitation spectrum; the method is a non-diagnostic treatment method.

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