CN109651495B - Orange fluorescent protein, nucleic acid molecule, vector, fusion protein and application - Google Patents

Abstract

The invention relates to the technical field of biomedical optics and molecular imaging, in particular to orange fluorescent protein, nucleic acid molecules, a vector, fusion protein and application. The orange fluorescent protein is one of the following sequences: a) as shown in SEQ ID NO: 1; b) as shown in SEQ ID NO: 1 by deletion, substitution or addition of one or more amino acid residues. The orange fluorescent protein has high brightness, strong monomer, larger Stokes displacement, strong tissue penetrability and good cell membrane positioning effect.

Description

Orange fluorescent protein, nucleic acid molecule, vector, fusion protein and application

Technical Field

The invention relates to the technical field of biomedical optics and molecular imaging, in particular to orange fluorescent protein, nucleic acid molecules, a vector, fusion protein and application.

Background

Fluorescent proteins (fluorescent proteins) have important applications in studying gene expression levels, protein interactions, protein conformations and protein activities in living cells in the fields of biomedical optics and molecular imaging. However, in the case of using fluorescent proteins to detect specific physiological activities to address biological problems of interest, it is often desirable that the fluorescent proteins have a number of specific properties.

The human brain contains about one billion neurons, which are connected with each other through axons and dendrites to form a huge signal transmission and storage neural network system. There are complex kinetic interactions between neurons. Signals from upstream neurons are transmitted in axons through action potentials to axon terminals in the form of membrane currents. The change of calcium ion concentration in synapses is caused, calcium ion signals regulate the fusion of synaptic vesicles and presynaptic membranes, chemical signal molecules such as neurotransmitters and glutamic acid are released to synaptic clefts, ligand gate ion channels on postsynaptic membranes of downstream neurons are opened, and the downstream neurons are triggered to generate nerve signals. Currently, gene-coded probes for detecting these neural activities mainly include calcium ion probes, membrane potential probes, pH probes and neurotransmitter probes, and fluorescent proteins have been widely used for the construction of these probes at the cellular and sub-cellular level. Among the above physicochemical changes in neural activity, the rapid transmission of action potential in axons, marked by a rapid change in membrane potential, reflects the nature of neural activity. Therefore, membrane potential probes are the most important class of nerve signal detection probes. The whole process from depolarization to repolarization of an action potential is about 1-3 milliseconds, which requires that the membrane potential probe has a response speed of the order of sub-milliseconds. At present, only an eFRET type membrane potential probe Ace2N-mNeonGreen based on rhodopsin protein and green fluorescent protein can really achieve sub-millisecond response to membrane potential change.

However, the technology that has just emerged in 2016 also has three significant limitations: 1. the dynamic range of the living body detection is low; 2. the fluorescent probe is a green fluorescent probe, has low tissue penetrability of fluorescence, and cannot be combined with optogenetics to carry out pure optical regulation and detection on nerve signals; 3. the green fluorescent protein mNeon Green used in the probe has poor two-photon performance and is not suitable for two-photon imaging. If the limitations are broken through, the detection dynamic range of the eFRET membrane potential probe is improved, the tissue penetrability of fluorescence of the eFRET membrane potential probe is improved, the performance of the membrane potential probe is greatly improved, and the development of neuroscience, particularly brain science, is promoted. An eFRET type membrane potential probe is a fusion protein constructed by using rhodopsin protein and fluorescent protein to form a pair of intramolecular FRET (fluorescence resonance energy transfer) pairs which are jointly expressed on neuron cell membranes. When the area where the probe is located is at a resting potential, FRET of the two proteins is weaker, and the fluorescent protein emits stronger fluorescence. When nerve current passes through the membrane, the membrane potential changes, the absorption spectrum of the rhodopsin protein changes, and strong FRET is generated between the rhodopsin protein and the fluorescent protein. At this time, the rhodopsin protein absorbs a large amount of light energy of photons emitted by the fluorescent protein, and the fluorescent protein becomes dark in brightness. This technique detects changes in membrane potential as a measure of changes in probe brightness, the dynamic range of which depends on two important factors: 1. FRET efficiency of both proteins. 2. The amount of rhodopsin protein in response to a change in membrane potential. The emission spectrum of the orange fluorescent protein is greatly overlapped with the absorption spectrum of the rhodopsin protein, so that the FRET efficiency can be well improved. At the same time, light of the wavelength of the excitation light of the orange fluorescent protein can also increase the amount of rhodopsin protein that responds to changes in membrane potential. Therefore, the construction of an eFRET membrane potential probe by replacing the green fluorescent protein with the orange fluorescent protein can improve the dynamic range of the probe. Furthermore, orange fluorescence scatters less in tissue than green fluorescence, and tissue penetration is higher. The orange fluorescent protein is used for replacing green fluorescent protein, so that two defects of the existing eFRET type membrane potential probe can be well overcome.

The orange fluorescent proteins most commonly used at present are the two series of mOrange and mKO. mOrange is an orange fluorescent protein that appeared in 2004 and is a derivative obtained by protein engineering of the red fluorescent protein DsRed (Shanner et al. improved monomeric red, orange and yellow fluorescent proteins derived from fluorescent Spectrum sp. red fluorescent protein. Nature Biotechnology 2004.22(12): 1567-1572.). It has poor light stability, low brightness and slow maturation. Shaner et al, university of California, 2008, optimized it to obtain a more photostable derivative designated mOrange2(Shaner et al, improvement of photostability of bright monomeric orange and red fluorescent proteins. Nature Methods2008.5 (6): 545-551.). The defects of low brightness (34.8) and slow maturation of the mOrage 2 are continued, but the mOrage 2 still has wide application due to rare orange fluorescent protein.

mKO kappa is the orange fluorescent protein with the best optical performance at present (Tsutsui et al, improvement membrane fluorescence using FRET with new fluorescent proteins 2008.5(8):683-685.), and the EC value of the mKO kappa reaches 105000cm^-1·mol^-1And the brightness reaches 64. However, monomers are poorly tolerated and when expressed in mammalian cells, accumulate in large numbers within the cell. mKO2, which belongs to the mKO series with mKO κ, was used for the study of the spatio-temporal dynamics of the Cell Cycle (Sakaue-Sawano et al, visualization Spatiometric dynamic porous multicell Cell-Cycle progression. Cell 2008.132(3): 487-498.). The optical performance of mKO2 was poor and the brightness was very low (39.6).

The excitation peak of the common orange fluorescent protein is about 550, while the excitation light of the current common two-photon laser is generally below 1000nm, which limits the wide application of the common orange fluorescent protein in the two-photon imaging technology. And these proteins have small stokes shifts (difference between the maximum emission wavelength and the maximum excitation wavelength of a fluorescent substance) and are all tens of nanometers. The emission peak is about 560nm, and the tissue penetrability is not good enough.

As can be seen, the currently available orange fluorescent proteins are very rare and are not suitable for constructing membrane potential probes: 1. the commonly used orange fluorescent protein mOrange2 has been proved to have poor cell membrane positioning effect when used for constructing a membrane potential probe, the probe is gathered in cytoplasm in a large amount, membrane application is difficult, and the brightness of the probe is low, so that the initial value of the brightness of the probe is greatly reduced. 2. The high-brightness mKO κ is not only poorly monomeric, but also undergoes extensive aggregation when expressed alone in cells. Therefore, the technical problems to be solved by the invention are as follows: a novel orange fluorescent protein with strong monomer property, high brightness and good cell membrane positioning effect is developed, and the problems are effectively solved.

Disclosure of Invention

In view of the above, the present invention provides orange fluorescent proteins, nucleic acid molecules, vectors, fusion proteins and uses. The orange fluorescent protein has high brightness, strong monomer, larger Stokes displacement, strong tissue penetrability and good cell membrane positioning effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides orange fluorescent protein, which is one of the following sequences:

a) as shown in SEQ ID NO: 1;

b) as shown in SEQ ID NO: 1 by deletion, substitution or addition of one or more amino acid residues.

The invention screens out a brand new orange fluorescent protein named mGeOFP1 (protein sequence shown in SEQ ID NO: 1), which has the characteristics of strong monomer, high brightness, good cell membrane positioning effect and the like. Its Stokes shift is greater than that of the normal orange fluorescent protein (40nm, 16nm for mOrange2 and 12nm for mKO κ). The single photon excitation peak is 532nm, and can be well excited by two-photon exciters of 710nm and 960-1000nm, and the emission peak is 578nm, so that the tissue penetrability is improved. The above characteristics can effectively improve the depth and sensitivity of in vivo imaging. mGeOFP1 can be well co-localized with rhodopsin mutant Ace2N of Fucus vesiculosus on the cell membrane of neuron and can be used for high-fidelity nerve electrical signal detection.

The invention also provides a nucleic acid molecule for encoding the orange fluorescent protein.

Preferably, the base sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 2, respectively. However, this base sequence is not a unique sequence, and all sequences encoding the nucleotide sequence of SEQ ID NO: 1 is within the scope of the present invention.

The invention also provides a vector comprising the nucleic acid molecule provided by the invention.

The invention also provides a host cell comprising the vector.

The invention also provides application of the orange fluorescent protein in protein localization or imaging.

The invention also provides application of the orange fluorescent protein in preparation of a membrane potential probe or two-photon imaging.

The invention also provides a fusion protein containing the orange fluorescent protein.

In particular embodiments provided herein, the fusion protein further comprises a protein of interest. The orange fluorescent protein can be used for positioning the target protein after being fused with the target protein, and the orange fluorescent protein does not interfere the positioning of the target protein in cells.

In another embodiment provided by the invention, the fusion protein further comprises a rhodopsin protein. The fusion protein membrane potential probe is constructed after the orange fluorescent protein and the rhodopsin protein are fused, the membrane location effect of the membrane potential probe is good, the membrane clamp and the membrane potential probe are used for simultaneously detecting electric signals and optical signals of the same neuron cell, and the membrane potential probe is found to generate high-fidelity rapid synchronous response to nerve signals. The response speed reaches the sub-millisecond level and is completely consistent with the recording frequency of the electric signals.

Preferably, the rhodopsin protein is an umbrella alga rhodopsin protein Ace or a mutant thereof.

The invention provides orange fluorescent protein, nucleic acid molecules, vectors, fusion protein and application. The orange fluorescent protein is one of the following sequences: a) as shown in SEQ ID NO: 1; b) as shown in SEQ ID NO: 1 by deletion, substitution or addition of one or more amino acid residues. The invention has the technical effects that:

the core of the invention lies in that a brand-new high-performance orange fluorescent protein mGeOFP1 with high brightness, strong monomer and larger Stokes displacement is screened out through mutation. The single photon excitation peak is 532nm, the single photon excitation peak can be well excited by two-photon exciters of 710nm and 960-1000nm, the emission peak is 578nm, the tissue penetrability is strong, and the depth and the sensitivity of in-vivo imaging can be effectively improved. The kit can construct a FRET pair with rhodopsin protein, perform fusion expression, co-locate on cell membranes of neurons, and perform high-fidelity detection of nerve electrical signals.

Drawings

FIG. 1 is a technical process for obtaining mGeOFP1 by protein engineering of mRuby 3; a 3D simulated complete structural drawing (left) and structural cross-sectional drawing (right) of mruby3 (PDB: 5 ltr); beta sheet forms a barrel-shaped structure to surround the middle luminous group, wherein the main chain is represented by cartoon format, and the luminous group is represented by stick format; green, yellow, cyan and magenta on the alpha helix and beta sheet to mark mutated amino acid sites around the luminophore; B. a schematic diagram of a fluorescence screening system; the excitation filter wheel, emission filter wheel and CCD in the figure can be controlled by a source software mu Manager; C. a schematic diagram of protein engineering design idea;

FIG. 2 shows the mutation site of mGeOFP1 and its performance characterization; graphs of excitation spectrum (orange thin line) and emission spectrum (orange thick line) of mgeofp1; mruby3 and mGeOFP1 protein amino acid sequence comparisons, with colored backgrounds indicating mutated amino acids; C. analyzing the monosomy of the mGeOFP1 by high performance liquid chromatography, wherein a black line represents a known dimeric fluorescent protein mKate2, a red line represents a known monomeric fluorescent protein fused Red, and an orange line represents mGeOFP1, wherein the peak emergence time of the mGeOFP1 is latest;

FIG. 3 is a graph of mGeOFP1 expression in HEK293T cells and two-photon spectroscopy; mgeofp1 is expressed in whole HEK293T cells; mGeOFP1-CAAX fusion protein is expressed on the cell membrane of HEK293T cells; mGeOFP1-H2B fusion protein is expressed in the nucleus of HEK293T cell, the cell is in the metaphase of mitosis, and homologous chromosome pairing can be observed; two-photon excitation spectrum (700-1000nm) of mGeOFP1; error bar: 10 mu m;

FIG. 4 shows the construction and performance detection of Ace2N-mGeOFP1 membrane potential probe; A. schematic diagrams of probe construction, membrane application and patch clamp addition; ace2n-mGeOFP1 was localized on neuronal cell membranes (grey scale pictures); C. the photoelectric signal synchronously records the action potential; the variation range of the electrical signal is 100mV, and the optical signal of the probe decreases with the increase of the membrane potential.

Detailed Description

The invention discloses orange fluorescent protein, nucleic acid molecule, vector, fusion protein and application, and can be realized by appropriately modifying process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The orange fluorescent protein, the nucleic acid molecule, the carrier, the fusion protein and the reagent or instrument used in the application are all available in the market.

The invention is further illustrated by the following examples:

example 1

The crystal structure analysis (figure 1A) is carried out on the red fluorescent protein mRuby3, the crystal structure analysis is compared with a homologous sequence, the reasonable design and site-directed mutation are carried out on key sites influencing the spectrum of the fluorescent protein and amino acids interacting with the key sites, then the mutant is expressed and screened on a constitutive expression vector pNCS, and the used expression strain is XL-10Gold (purchased from Agilent technologies). To ensure the integrity of the library, 10 clones were set for each mutant, and finally the fluorescent properties of the mutants were detected by visual discrimination and blue LED excitation light through an orange acrylic filter, and a single clone expressing a spectrum blue-shifted fluorescent protein was screened (fig. 1B is a schematic diagram of a fluorescence screening apparatus). And then carrying out site-directed mutagenesis on non-conservative amino acid residues around the luminescent group of the mutant fluorescent protein with the spectrum blue shift, stabilizing the luminescent group, screening out a monoclonal with high quantum yield (high brightness), and naming the monoclonal as mGeOFP1, wherein the amino acid sequence of the monoclonal is shown in SEQ ID NO: 1:

MVSKGEELIKEEMPMKVVMTGTVNGHYFKCTGEGEGRPYEGVQTQRIKVIEGGPLPFAFDILSTSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRFEDGGVLTSTQDTSLQDGVLIYNVKVRGENFPSNGPVMQKKTKGWEPSTEMMYPADGGLRGYVDKALKVDGGGHLHCSFVTEYKSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVAKYSNLGGGMDELYK

the gene sequence for coding the protein is SEQ ID NO: 2:

ATGGTGTCTAAGGGCGAAGAGCTGATCAAGGAAGAGATGCCCATGAAGGTGGTCATGACAGGTACCGTCAACGGCCACTACTTCAAGTGCACAGGCGAAGGAGAAGGCAGACCGTACGAGGGAGTGCAAACCCAGAGGATCAAAGTCATCGAGGGAGGACCCCTGCCATTTGCCTTTGACATTCTTAGCACGTCGTTCATGTATGGCAGCCGTACCTTTATCAAGTACCCGGCCGACATCCCTGATTTCTTTAAACAGTCCTTTCCTGAGGGTTTTACTTGGGAAAGAGTTACGAGATTCGAAGATGGTGGAGTCCTCACCAGCACGCAGGACACCAGCCTTCAGGATGGCGTGCTCATCTACAACGTCAAGGTCAGAGGGGAGAACTTTCCCTCCAATGGTCCCGTGATGCAGAAGAAGACCAAGGGTTGGGAGCCCTCCACAGAGATGATGTATCCAGCAGATGGTGGTCTGAGAGGATACGTCGACAAGGCACTGAAAGTTGATGGTGGTGGCCATCTGCACTGCAGCTTCGTGACAGAATACAAGTCAAAAAAGACCGTCGGGAACATCAAGATGCCCGGTGTCCATGCCGTTGATCACCGCCTGGAAAGGATCGAGGAGAGTGACAATGAAACCTACGTAGTGCAAAGAGAAGTGGCAGTTGCCAAATACAGCAACCTTGGTGGTGGCATGGACGAGCTGTACAAG

the general protein engineering design concept is schematically shown in figure 1C. Sequencing results showed that mGeOFP1 mutated 21 sites based on mRuby3 (FIG. 2B).

Example 2

The bacteria expressing mGeOFP1 were lysed using B-PER II (purchased from Pierce Inc.), thenThe protein was then purified using HisPur Cobalt Resin (purchased from Pierce), followed by desalting through Econo-Pac10DG gravity flow chromatography (purchased from Bio-Rad, USA). After completion of the above protein purification steps, the single photon excitation and emission spectra of mGeOFP1 were examined using Lambda35UV/VIS and LS-55 fluorescence spectrometer (purchased from Perkin Elmer). As shown in Table 1 and FIG. 2A, the Stokes shift of mGeOFP1 was 40nm, which is larger than that of the ordinary orange fluorescent protein. The excitation peak is 532nm, which is blue-shifted compared with other orange fluorescent proteins, and is easier to be excited by two-photon excitation light with the wavelength less than 1000 nm. And the emission peak is 578nm, and compared with other orange fluorescent proteins, the red shift is stronger in tissue penetrability. Another difference is that the extinction coefficient of mGeOFP1 reaches 91331cm^-1·mol^-1Higher than most orange fluorescent proteins.

TABLE 1 optical Properties of mGeOFP1 in comparison with other orange fluorescent proteins for the corresponding functions of different mutation sites

Example 3

The purified mGeOFP1 protein was concentrated to a high concentration of 10mg/mL and chromatographed using high performance liquid chromatography (Shimadzu LC20A) to detect the monosomy of the protein. As shown in fig. 2C, the peak time of mGeOFP1 was 32.59 minutes while the known monomeric fluorescent protein fusion red was 32.38 minutes, with a later peak time indicating a stronger monomer, indicating that mGeOFP1 has better monomer than fusion red.

Example 4

The purified mGeOFP1 protein was concentrated to a high concentration of 5mg/mL, and the readings of fluorescence emitted by mGeOFP1 when excited by excitation light of the same wavelength in buffers of different pH values were examined using Lambda35UV/VIS and LS-55 fluorescence spectrometer (purchased from Perkin Elmer Co.), and the pKa value of the protein was calculated therefrom. As shown in table 1, mGeOFP1 has a pKa of 3.7, which is lower than that of other known orange fluorescent proteins, indicating that mGeOFP1 has better optical properties and is more resistant to acidic environments in a more acidic environment than other orange fluorescent proteins.

Example 5

The two-photon excitation spectrum of mGeOFP1 below 1000nm was examined using an IX81 commercial microscope (available from olympus) coupled with a two-level titanium sapphire laser (available from coherent). As shown in FIG. 3D, mGeOFP1 is well excited by two photons both at 710nm and at 960-1000 nm.

Example 6

The plasmid containing the mGeOFP1 gene and the CAG promoter is transfected into HEK293T cells by a Lipofectamine 2000 kit, pmGeOFP1-CAAX and pmGeOFP1-H2B are constructed on the basis of an expression vector pEGFP-C1, and the expression of mGeOFP1 in mammalian cells and the positioning condition of cell membranes and cell nuclei are observed. As shown in FIGS. 3A, B and C, mGeOFP1 still has better fusion with other proteins at different subcellular positions, which indicates that mGeOFP1 does not interfere with the intracellular localization of the protein to be studied.

Example 7

A fusion protein membrane potential probe (shown in figure 4A) is constructed by rhodopsin protein mutants Ace2N and mGeOFP1 of the umbrella algae, and is transfected into primary cultured neuronal cells through a lentiviral vector pLenti-CamkII and is expressed, so that the cell membrane localization effect of Ace2N-mGeOFP1 is good (shown in figure 4B). The patch clamp and the Ace2N-mGeOFP1 probe are used for simultaneously detecting electric signals and optical signals of the same neuron cell, and the probe can generate high-fidelity rapid synchronous response to neural signals. The response speed reaches the sub-millisecond level and is completely consistent with the recording frequency of the electric signal (figure 4C).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims (10)

1. An orange fluorescent protein, which is characterized in that the orange fluorescent protein is shown as SEQ ID NO: 1.

2. A nucleic acid molecule encoding the orange fluorescent protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the base sequence of said nucleic acid molecule is as set forth in SEQ id no: 2, respectively.

4. A vector comprising the nucleic acid molecule of claim 2 or 3.

5. A host cell comprising the vector of claim 4.

6. Use of the orange fluorescent protein according to claim 1 for protein localization or protein imaging.

7. Use of the orange fluorescent protein according to claim 1 for the preparation of a membrane potential probe or two-photon imaging.

8. A fusion protein comprising the orange fluorescent protein of claim 1.

9. The fusion protein of claim 8, further comprising a protein of interest.

10. The fusion protein of claim 8, further comprising a rhodopsin protein.

Images