JP7613726B2 - Method for producing peptides and cAMP indicators - Google Patents

Description

The present invention relates to a cAMP indicator for real-time imaging of the dynamic behavior of cAMP and a method for producing the same.

Many hormones and neurotransmitters act on G protein-coupled receptors, triggering various cellular activities via intracellular second messengers. Second messengers are molecules that relay signals received on the cell surface. Protein hormones, growth factors, etc. reach receptors on the cell surface, and the second messengers receive them and act on molecules in the cytoplasm or nucleus. Second messengers not only relay signals, but also amplify them.

Typical second messengers include cyclic AMP (hereinafter sometimes referred to as cAMP), cyclic GMP (hereinafter sometimes referred to as cGMP), inositol phosphate, diacylglycerol, calcium ions, etc. In G protein-coupled receptors, different second messengers are produced depending on the G protein that binds to the receptor. For example, in receptors that couple with Gs proteins (such as the beta-adrenergic receptor and glucagon receptor), adenylate cyclase is activated and cAMP is produced.

Genetic expression-type cAMP indicators that have been reported at the basic research level include R-FlincA (Red Fluorescent indicator for cAMP), Pink-Flamindo, Flamindo2, CAMPr, ICUE, EPAC-S, and EPAC-cAMP. One commercially available product is cADDis.

R-FlincA is a highly sensitive red fluorescent cAMP indicator that uses the fluorescent protein mApple. By using R-FlincA, whose brightness increases upon binding to low concentrations of cAMP, it is possible to detect the dynamics of low concentrations of cAMP (Non-Patent Document 1).

Flamindo2 is a cAMP sensor based on green fluorescent protein. It is called a monochromatic brightness change sensor, and has a structure in which the binding site for the target molecule is linked between the divided fluorescent protein. It is thought that when the target molecule binds, the structure of the fluorescent protein changes, causing a change in the fluorescence intensity (Non-Patent Document 2).

cAMP is involved in regulating a variety of physiological functions; for example, in pancreatic β cells, an increase in intracellular cAMP concentration promotes insulin secretion, and in neurons in the hippocampus of the brain, an increase in cAMP concentration induces a memory learning response. A highly specific indicator is needed to analyze the intracellular dynamics of cAMP in these various physiological functions.

However, no indicator with a sufficient range of reaction change has been developed that is more specific to cAMP than to cGMP.

Yusaku Ohta et al.,Scientific Reports, 8(1866)1-9, Published online 30 January 2018 Kitaguchi et al., Biochem J, 2013; Odaka et al., PLoS ONE, 2014

The present invention was made in consideration of these problems, and aims to provide a highly specific cAMP indicator and a method for producing the same.

The cAMP indicator of the present invention is a cAMP indicator for real-time imaging of the dynamic behavior of cAMP in cells using green excitation light, and is characterized by having the amino acid sequence shown in SEQ ID NO:1.

The cAMP indicator of the present invention is a cAMP indicator for real-time imaging of the dynamic behavior of cAMP in cells using violet excitation light, and is characterized by having the amino acid sequence shown in SEQ ID NO:2.

The method for producing a cAMP indicator according to the present invention is characterized by comprising a step of introducing DNA into cells using a vector having the DNA sequence set forth in SEQ ID NO:3.

The method for producing a cAMP indicator according to the present invention is characterized by comprising a step of introducing DNA into cells by using a vector having the DNA sequence set forth in SEQ ID NO:4.

The present invention provides a highly specific cAMP indicator.

FIG. 1 is a graph showing that the cAMP indicator of the present invention has higher specificity for cAMP than for cGMP. FIG. 1 shows the excitation spectrum and fluorescence spectrum of gCarvi protein. FIG. 1 shows the excitation spectrum and fluorescence spectrum of gvCarvi protein. Photographic diagram showing the expression of gCarvi protein in hippocampal neurons.

The following describes in detail an embodiment of the present invention with reference to the attached drawings. However, this embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiment. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiment are also included in the scope of the present invention.

The inventors conducted screening while checking the cAMP/cGMP ratio based on a cAMP indicator that utilizes the cAMP binding domain of the E. coli cAMP receptor protein, and discovered a cAMP indicator with high cAMP specificity, completing the present invention based on this fact. The cAMP indicator of the present invention is sometimes called Carvi. Carvi that can be imaged with green excitation light is sometimes called gCarvi. Carvi that can be imaged with violet excitation light is sometimes called gvCarvi.

The cAMP indicator (gCarvi) of the present invention is characterized by having the amino acid sequence set forth in SEQ ID NO:1.

The cAMP indicator (gCarvi) of the present invention is characterized by having a sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 1. This is because mutations in an amino acid sequence that is 90% or more identical may not inhibit the functional identity of the protein.

The cAMP indicator (gCarvi) of the present invention is characterized in that it has a sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO:1. This is because the identity of the protein's function may not be inhibited in the case of mutations in one or more amino acid sequences.

The wavelength of the green excitation light is not particularly limited, but is, for example, 490 to 550 nm.

The cAMP indicator (gvCarvi) of the present invention is characterized by having the amino acid sequence shown in SEQ ID NO:2.

The cAMP indicator (gvCarvi) of the present invention is characterized by having a sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:2.

The cAMP indicator (gvCarvi) of the present invention is characterized in that it has an amino acid sequence represented by SEQ ID NO:2 in which one or more amino acids have been substituted, deleted, inserted or added.

The wavelength of the violet excitation light is not particularly limited, but is, for example, 380 to 430 nm.

The cells to be observed for the dynamic behavior of cAMP are not particularly limited, but may be, for example, nerve cells. The nerve cells are not particularly limited, but may be nerve cells in tissues or organs that constitute the nervous system, such as the central nervous system, such as the cerebral cortex (neocortex, limbic cortex such as the hippocampus), diencephalon (thalamus, hypothalamus, basal ganglia, etc.), cerebellum, pons, medulla oblongata, and spinal cord, or the peripheral nervous system distributed on the body surface, deep tissues, and internal organs, and are preferably hippocampal nerve cells.

cAMP is a type of cyclic nucleotide in which the 3' and 5' positions of adenosine ribose and phosphate are cyclically linked, and mediates a wide variety of physiological responses of cells in response to extracellular ligands. cAMP is synthesized from adenosine triphosphate (ATP) in the cytoplasm by the action of adenylate cyclase, and is rapidly decomposed to adenosine 5'-AMP by the action of cyclic nucleotide phosphodiesterase (PDE). Three well-known cAMP effector molecules are protein kinase A (PKA), Epac, and CNG channel, which mediate a wide variety of cAMP downstream signals. In nerve cells, cAMP is involved in a wide variety of processes, such as differentiation, survival, polarity formation, process extension, axon guidance, axon regeneration, synaptic transmission, synaptic plasticity, and hormone secretion. Therefore, the effect of the present invention, which allows specific imaging of cAMP, is important.

The method for producing the cAMP indicator (gCarvi) of the present invention is characterized by comprising a step of introducing the DNA into cells by using a vector having the DNA sequence set forth in SEQ ID NO:3.

The wavelength of the green excitation light is not particularly limited, but is, for example, 490 to 550 nm.

The method for producing the cAMP indicator (gvCarvi) of the present invention is characterized by comprising a step of introducing the DNA into cells using a vector having the DNA sequence set forth in SEQ ID NO:4.

The wavelength of the violet excitation light is not particularly limited, but is, for example, 380 to 430 nm.

The vector is not particularly limited, but may be, for example, an E. coli expression vector.

(1) Obtaining gCarvi and gvCarvi DNA sequences
The sequence of the cAMP binding region was obtained by PCR using the genomic DNA of E. coli (DH5α strain) as a template. The PCR primers were as follows:
Forward primer: TAGGATCCATGGTGCTTGGCAAACCG (SEQ ID NO:5)
Reverse primer: TTACTCGAGCGCCAGGTTGCCCAC (SEQ ID NO: 6)
Using the obtained cAMP binding region sequence as a template, a cAMP binding region DNA sequence for Carvi was obtained by PCR in which the 135th amino acid of CRP (cAMP receptor protein) was replaced with tyrosine. The PCR primers are as follows:
Forward primer: TAGGATCCATGGTGCTTGGCAAACCG (SEQ ID NO: 7)
Reverse primer: TTACTCGAGATACAGGTTGCCCAC (SEQ ID NO:8)
The cpGFP (a circularly permuted variant of the fluorescent protein GFP) sequence was obtained by PCR using the DNA sequence of GCaMP3 (a calcium sensor protein) as a template. The PCR primers are as follows:
Forward primer: TAGGATCCCTCGAGAACGTCTATATCAAGGC (SEQ ID NO: 9)
Reverse primer: TAAGTCGACAAGCTTACTAGTGTTGTACTCCAGCTTGTGCC (SEQ ID NO: 10)
Using the obtained cpGFP sequence as a template, the cpGFP DNA sequence for gCarvi was obtained by PCR in which the 58th amino acid of cpGFP was replaced with isoleucine and the 112th amino acid of cpGFP was replaced with glutamine. The PCR primers are as follows:
Sense primer #1: CGTGCAGTCCATTCTTTCGAA (SEQ ID NO: 11)
Antisense primer #1: TTCGAAAGAATGGACTGCACG (SEQ ID NO: 12)
Sense primer #2: GCCCATCCAGGTCGAGC (SEQ ID NO: 13)
Antisense primer #2: GCTCGACCTGGATGGGC (SEQ ID NO: 14)
Using the obtained cpGFP DNA sequence for gCarvi as a template, the cpGFP DNA sequence for gvCarvi was obtained by PCR in which the 8th amino acid of cpGFP was replaced with glutamic acid and the 162nd amino acid of cpGFP was replaced with serine. The PCR primers are as follows:
Sense primer #3: AAGGCCGACGAGCAGAAGAAC (SEQ ID NO: 15)
Antisense primer #3: GTTCTTCTGCTCGTCGGCCTT (SEQ ID NO: 16)
Sense primer #4: CACCCTGTCCTACGGCGT (SEQ ID NO: 17)
Antisense primer #4: ACGCCGTAGGACAGGGTG (SEQ ID NO: 18)
The cAMP binding region DNA sequence for Carvi and the cpGFP DNA sequence for gCarvi were cut at the restriction enzyme Xho I site (meaning that the [5'-CTCGAG-3'] sequence is cut to [C/TCGAG]. The same applies below.) and then ligated to form the gCarvi sequence. The cAMP binding region DNA sequence for Carvi and the cpGFP DNA sequence for gvCarvi were cut at the restriction enzyme Xho I site and then ligated to form the gvCarvi sequence.

(2) Production of gCarvi purified protein
gCarvi is a cAMP indicator according to the present invention that performs imaging using green excitation light of 490 to 550 nm. The gCarvi DNA sequence according to SEQ ID NO: 3 was incorporated into the E. coli expression vector pCold I (Takara Bio). pCold I-Carvi was transformed into E. coli BL21 strain, and gCarvi protein expression was induced according to the method in accordance with the pCold I manual (15°C, 24 hours, 0.5 mM IPTG added). Then, gCarvi protein consisting of the amino acid sequence shown in SEQ ID NO: 1 was isolated and purified using cobalt resin, utilizing the histidine tag sequence contained in the pCold I sequence.

(3) Production of gvCarvi purified protein
gvCarvi is a cAMP indicator according to the present invention that performs imaging using purple excitation light of 380 to 430 nm. The gvCarvi DNA sequence according to SEQ ID NO: 4 was incorporated into the E. coli expression vector pCold I (Takara Bio). pCold I-Carvi was transformed into E. coli BL21 strain, and gvCarvi protein expression was induced according to the method in accordance with the pCold I manual. Then, the gvCarvi protein consisting of the amino acid sequence described in SEQ ID NO: 2 was isolated and purified using cobalt resin, utilizing the histidine tag sequence contained in the pCold I sequence.

(4) Expression of gCarvi protein in cultured hippocampal neurons The gCarvi DNA sequence shown in SEQ ID NO: 3 was incorporated into pAAV2-hSyn-TetOFF, a mammalian neuronal cell-specific expression vector. AAV-Carvi was produced by transfecting HEK293T cells with pAAV2-hSyn-TetOFF-Carvi, pRC1 (Takara Bio), and pHelper (Takara Bio). By infecting hippocampal neurons on the 3rd to 6th day of culture with AAV-Carvi, the expression of gCarvi protein consisting of the amino acid sequence shown in SEQ ID NO: 1 can be confirmed from the 12th day of culture onwards.

(5) Expression of gvCarvi protein in cultured hippocampal neurons The gvCarvi DNA sequence shown in SEQ ID NO: 4 was incorporated into pAAV2-hSyn-TetOFF, a mammalian neuronal cell-specific expression vector. AAV-Carvi was produced by transfecting HEK293T cells with pAAV2-hSyn-TetOFF-Carvi, pRC1 (Takara Bio), and pHelper (Takara Bio). By infecting hippocampal neurons on the 3rd to 6th day of culture with AAV-Carvi, the expression of gvCarvi protein consisting of the amino acid sequence shown in SEQ ID NO: 2 can be confirmed on or after the 12th day of culture.

(6) cAMP specificity of gCarvi protein Purified gCarvi protein was dissolved in 50 mM HEPES buffer and dispensed into a 96-well black plate (Thermo Scientific #237108). The change in fluorescence intensity upon addition of cAMP (Sigma) or cGMP (Sigma) was measured using a microplate reader (Tecan, infinite F200).

As shown in Figure 1, the specificity for cAMP over cGMP became more pronounced at around 1 μM, and the specificity for cAMP was prominent at around 10 μM.

(7) Excitation and fluorescence spectra of gCarvi protein Purified gCarvi protein was dissolved in 50 mM HEPES buffer, and its excitation and fluorescence spectra were analyzed using a spectrometer (Shimadzu, UV-1800). The dotted line shows the spectrum of gCarvi alone, and the dashed and solid lines show the spectrum when 3 μM and 10 μM cAMP were added, respectively.

As shown in Figure 2, the excitation spectrum and fluorescence spectrum of the gCarvi protein were nearly overlapping, making it possible to image the dynamic behavior of cAMP in real time using existing experimental equipment.

(8) Excitation and fluorescence spectra of gvCarvi protein Purified gvCarvi protein was dissolved in 50 mM HEPES buffer, and its excitation and fluorescence spectra were analyzed using a spectrometer (Shimadzu, UV-1800). The dotted line shows the spectrum of gvCarvi alone, and the dashed and solid lines show the spectrum when 1 μM and 10 μM cAMP were added, respectively.

As shown in Figure 3, the gvCarvi protein uses violet excitation light, which has not been used before, and the excitation spectrum and fluorescence spectrum of the gvCarvi protein do not overlap. Therefore, it is possible to image the dynamic behavior of cAMP in real time while coexisting with an experimental system using violet excitation light.

(9) Expression of gCarvi protein in hippocampal neurons
By infecting hippocampal neurons on the 3rd to 6th day of culture with AAV-Carvi, the expression of gCarvi protein can be confirmed from the 12th day of culture onwards. Fluorescence images (Fig. 4, left and middle) and differential interference images (Fig. 4, right) of gCarvi protein expressed in hippocampal neurons are shown. Adenylate cyclase is an enzyme that produces cAMP, and forskolin is an agonist of adenylate cyclase. Before forskolin administration, the cAMP concentration was low and the fluorescence of gCarvi was weak (Fig. 4, left). After forskolin administration, the cAMP concentration increased and the fluorescence of gCarvi became stronger (Fig. 4, middle). The morphology of gCarvi-expressing neurons was well developed, and no cytotoxicity of gCarvi was observed (Fig. 4, right).

Can be used for real-time imaging of cAMP in living cells.

SEQ ID NOs: 1-2: cAMP indicator SEQ ID NOs: 3-4: DNA to be carried in vector
SEQ ID NOs: 5 to 18: Primers

Claims (14)

A peptide that binds to cAMP and is used for imaging the dynamic behavior of cAMP in a cell using green excitation light, the peptide being characterized in that it is either (i) or (ii) below:
(i) a peptide having the amino acid sequence set forth in SEQ ID NO:1
(ii) a peptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:1 The peptide according to claim 1, characterized in that the wavelength of the green excitation light is 490 to 550 nm. The peptide according to claim 1 or 2, characterized in that the cell is a neuron. The peptide according to claim 3, characterized in that the neuron is a hippocampal neuron. A peptide that binds to cAMP and is used for imaging the dynamic behavior of cAMP in a cell using violet excitation light, the peptide being characterized in that it is either (i) or (ii) below:
(i) a peptide having the amino acid sequence set forth in SEQ ID NO:2
(ii) a peptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:2 The peptide according to claim 5, characterized in that the wavelength of the violet excitation light is 380 to 430 nm. The peptide according to claim 5 or 6, characterized in that the cell is a neuron. The peptide according to claim 7, characterized in that the neuron is a hippocampal neuron. A method for producing a cAMP indicator comprising the peptide according to any one of claims 1 to 4, which is imaged with green excitation light, the method comprising the step of introducing the DNA into cells by using a vector having the DNA sequence shown in SEQ ID NO:3. The method for producing a cAMP indicator according to claim 9, characterized in that the wavelength of the green excitation light is 490 to 550 nm. The method for producing a cAMP indicator according to claim 9 or 10, characterized in that the vector is an E. coli expression vector. A method for producing a cAMP indicator comprising the peptide according to any one of claims 5 to 8, which is imaged using violet excitation light, the method comprising the step of introducing the DNA into cells by using a vector having the DNA sequence shown in SEQ ID NO:4 . The method for producing a cAMP indicator according to claim 12, characterized in that the wavelength of the violet excitation light is 380 to 430 nm. The method for producing a cAMP indicator according to claim 12 or 13, characterized in that the vector is an E. coli expression vector.

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