KR20210029674A - Flavin mononucleotide binding protein variants derived from Pseudomonas putida with enhanced fluorescence intensity - Google Patents

Abstract

The present invention relates to a flavin-based fluorescent protein variant having improved fluorescence intensity. The present invention also relates to a gene encoding a flavin-based fluorescent protein variant. The present invention relates to a technology that can overcome the limitations of conventional detection systems using fluorescent proteins, which have different fluorescence intensity depending on the presence and concentration of oxygen, and the limitations of fluorescent proteins using FMN cofactors, which are difficult to use due to low fluorescence signal. Thus, the present invention can be widely used in various biotechnology fields such as life science research, biosensors, biochips, disease diagnosis, and the like.

Description

Flavin mononucleotide binding protein variants derived from Pseudomonas putida with enhanced fluorescence intensity}

The present invention relates to a flavin mononucleotide-binding fluorescent protein variant with improved fluorescence intensity, and to discovery of a fluorescent protein variant derived from Pseudomonas putida.

Fluorescent proteins are widely applied in various research fields as a reporter that can be encoded in genes for life science, medicine, and pharmaceutical research. In particular, the expression, activity, and secretion of proteins in cells, animals, and plants can be monitored in real time. It is usefully used for analysis. Fluorescent protein is a protein that emits light of a different wavelength in the process of being activated by light of a specific wavelength and then changing to a low energy level. As a representative example, GFP (Green fluorescent protein) was found in jellyfish living in the sea, and is a bioscience research. It has been used as an essential tool for research, and the Nobel Prize was awarded in 2008 for its technical research.

By linking a gene that generates GFP to a protein gene, the expression of the gene in cells or tissues can be tracked using GFP fluorescence, and the gene of the green fluorescent protein is inserted into the gene of a specific protein that causes cancer. When injected into cells, the size and location of the cancer can be tracked over time, so it can be visually confirmed by shining blue light without the need to dissect the organism.

In addition, in medical protein research, if a green fluorescent protein gene is fused to a target protein gene and inserted into an animal, it is also possible to easily determine whether the target protein is inserted through the green fluorescent protein.

In addition, bioscience studies such as analysis of neural circuits, cell membrane studies, and viral infection mechanisms are being actively conducted through GFP gene binding, and development of fluorescent animals introducing GFP into live animals has also been reported.

Most of the fluorescent proteins, including GFP that are currently used, have a limitation in that oxygen is essential to form a fluorescent chromophore capable of emitting fluorescent light. Therefore, it is difficult to use in an oxygen-deficient intracellular environment, anaerobic bacteria that maintain survival in oxygen-free conditions, and an environment lacking oxygen due to excessive growth of cells.

On the other hand, flavin-based fluorescent proteins (FbFPs), which can be used as fluorescent markers for life science, medicine, and pharmacy, have been discovered regardless of the presence of oxygen or oxygen deficiency. Flavin-based fluorescent proteins (FbFPs) have a disadvantage in that they are difficult to use because their fluorescence intensity is not high.

Matters described as the above-described background art are only for enhancing an understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

The present inventors made diligent efforts to develop a flavin-based fluorescent protein (FbFP) having a high fluorescence intensity. As a result, the present invention was completed by discovering a fluorescent protein variant having a significantly improved fluorescence intensity compared to the existing flavin-based fluorescent protein through a search for a large library in which the flavin-based fluorescent protein was mutated through random mutation.

Accordingly, an object of the present invention is to provide a flavin-based fluorescent protein variant with improved fluorescence intensity.

Another object of the present invention is to provide a gene encoding the flavin-based fluorescent protein variant.

Another object of the present invention is to provide an expression vector containing the gene.

Another object of the present invention is to provide a cell containing the gene or the expression vector.

Another object of the present invention is to provide a recombinant vector comprising the gene of the fluorescent protein variant as a reporter and comprising the gene encoding the target protein.

Another object of the present invention is to provide a cell containing the recombinant vector.

Another object of the present invention is to provide a method for analyzing the expression of a protein of interest by culturing the cells.

Another object of the present invention is to provide a method for separating and purifying a protein of interest by culturing the cells.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a flavin-based fluorescent protein variant (Flavin-based fluorescent protein, FbFP) with improved fluorescence intensity.

As a result of the present inventors' efforts to develop flavin-based fluorescent proteins with high fluorescence intensity, the fluorescence intensity was significantly improved compared to existing flavin-based fluorescent proteins by searching for a large library in which flavin-based fluorescent proteins were mutated through random mutation. Fluorescent protein variants were discovered.

According to a preferred embodiment of the present invention, the FbFP and FbFP variants are derived from Pseudomonas putida.

According to a preferred embodiment of the present invention, the FbFP variant includes a part of the amino acid sequence of FbFP of SEQ ID NO: 1, and H (Histidine), which is the 13th amino acid of the amino acid sequence of FbFP of SEQ ID NO: 1, is Y (Tyrosine); The 23rd amino acid, Q (Glutamine), is P (Proline), and the 53rd amino acid, C (Cysteine), is G (Glycine); 108th amino acid Q (Glutamine) to R (Arginine); 112th amino acid Y (Tyrosine) to F (Phenylalanine); 115th amino acid I (Isoleucine) to T (Threonine); And V (Valine), which is the 129th amino acid, is substituted with M (Methionine).

According to a preferred embodiment of the present invention, the FbFP variant is E (Glutamate), which is the 22nd amino acid of the amino acid sequence of FbFP of SEQ ID NO: 1 as K (Lysine); The 47th amino acid, D (Aspartate), is E (Glutamate); 54th amino acid R (Arginine) to S (Serine); And the 100th amino acid T (Threonine) further includes those substituted with A (Alanine).

According to a preferred embodiment of the present invention, the FbFP variant further includes a substitution of N (Asparagine) for D (Aspartate), which is the 107th amino acid in the amino acid sequence of FbFP in SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the FbFP variant additionally includes the one in which L (Leucine), which is the 86th amino acid in the amino acid sequence of FbFP of SEQ ID NO: 1, is substituted with I (Isoleucine).

According to a preferred embodiment of the present invention, the FbFP variant comprises an amino acid sequence selected from the group consisting of the second sequence, the third sequence, the fourth sequence, and the fifth sequence.

The FbFP variant of the present invention is 100% or more, preferably 500% or more, more preferably 800% or more, even more preferably 1000% or more compared to the FbFP protein (SB2) derived from Pseudomonas putida. It is characterized in that the fluorescence intensity is increased.

According to an embodiment of the present invention, the FbFP variants of the present invention exhibited a luminescence pattern that was increased by 10 to 11 times or more compared to the wild-type FbFP protein (SB2) (FIG. 5).

The FbFP variants of the present invention also show an increased quantum yield compared to the wild-type FbFP protein (SB2).

Quantum yield is a value representing the ratio of the number of photons or the number of emitted photons to the number of absorbed photons in light emission or photoelectron emission following absorption of light. In other words, the higher the quantum yield, the higher the conversion rate of absorbed light photons into photons that are emitted again. Therefore, a protein with a high quantum yield indicates that it is a bright fluorescent protein. Fluorescent proteins with high quantum yield can be measured to have relatively high fluorescence even when there is a small amount, which leads to an increase in sensitivity.

The FbFP variant of the present invention is characterized by an increase of 4% or more, preferably 100% or more, more preferably 500% or more, even more preferably 800% or more compared to wild-type FbFP protein (SB2).

According to an embodiment of the present invention, the FbFP variants of the present invention exhibited a 7- to 12-fold increase in quantum yield compared to the wild-type FbFP protein (SB2) (FIG. 6).

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a flavin-based fluorescent protein variant (FbFP).

According to another aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule.

According to another aspect of the present invention, the present invention provides a host cell comprising the nucleic acid molecule or the vector.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule as a reporter and comprising a gene encoding a protein of interest to be expressed.

According to another aspect of the present invention, the present invention provides a host cell comprising the recombinant vector.

The nucleic acid molecule of the present invention may be isolated or recombinant, and includes single-stranded and double-stranded DNA and RNA as well as corresponding complementary sequences. An “isolated nucleic acid”, in the case of a nucleic acid isolated from a naturally occurring source, is a nucleic acid isolated from surrounding genetic sequences present in the genome of an individual from which the nucleic acid was isolated. In the case of nucleic acids enzymatically or chemically synthesized from the template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acid resulting from this procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when expressed as a preprotein, which is a form before the polypeptide is secreted, and the promoter or enhancer is a polypeptide sequence. Is operably linked to the coding sequence when it affects the transcription of, or the ribosome binding site is operably linked to the coding sequence when arranged to facilitate translation. In general, "operably linked" means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, it means that they are contiguous and exist within the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at a convenient restriction enzyme site. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

In the present specification, the term "vector" refers to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences can be exogenous or heterologous. Examples of the vector include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophage, AAV, etc.). Those skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current. Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994, etc.).

As used herein, the term "expression vector" refers to a vector including a nucleic acid sequence encoding at least a portion of the gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. Along with the regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also provide other functions.

According to a preferred embodiment of the present invention, the vector includes the nucleic acid molecule as a reporter gene, and further includes a protein of interest to be expressed.

In the present specification, the term "target protein" or "target protein" means a protein to be identified or produced using an appropriate host cell.

When the nucleic acid molecule of the present invention is used as a reporter gene, the expression of the target protein can be analyzed by fluorescence analysis of the expression of the target protein, or the target protein can be produced and used for easy separation and purification.

In the present specification, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which means a process in which exogenous nucleic acid molecules are transferred or introduced into the host cell.

The host cells of the present invention are preferably bacterial cells, yeast, mammalian cells (CHO cells, HeLa cells, HEK293 cells, BHK cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells, MDCK cells). , WI38 cells, etc.), but is not limited thereto.

According to another aspect of the present invention, the present invention provides a composition for fluorescent coloring comprising the FbFP variant, nucleic acid molecule or vector.

The composition for generating fluorescence may be prepared by being included in a kit for confirming the expression of a target protein, separating and purifying the expressed target protein, or imaging a specific tissue, cell, or molecule.

According to another aspect of the present invention, the present invention provides a method for analyzing the expression of a protein of interest comprising the step of expressing the vector.

According to another aspect of the present invention, the present invention comprises the steps of generating a protein of interest by expressing the vector; And separating the generated target protein.

As described above, when the nucleic acid molecule of the present invention is used as a reporter gene, it can be used to confirm the expression of the target protein or to isolate and purify the expressed target protein, and the FbFP variant of the present invention is bound to another protein or molecule, or It can be used to identify or quantify their location by fusion and fluorescence.

According to another aspect of the present invention, the present invention provides a method for preparing an FbFP variant comprising the following steps:

a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the FbFP variant; And

b) recovering the FbFP variant expressed by the host cell.

According to another aspect of the present invention, the present invention provides a method for screening FbFP variants comprising the following steps:

a) constructing a library of a FbFP variant or a nucleic acid molecule encoding the FbFP variant or a nucleic acid molecule encoding the FbFP variant by adding a random point mutation to the nucleic acid molecule encoding the FbFP variant; And

b) selecting an FbFP variant or a nucleic acid molecule encoding the FbFP variant with increased fluorescence intensity than that of the FbFP protein of SEQ ID NO: 1 in the library.

The screening method of the present invention may use fluorescently labeled cell separation (FACS) screening or flow cytometry. Instruments for carrying out flow cytometry are known to those skilled in the art. Examples of such devices include FACSAria, FACS Star Plus, FACScan and FACSort instruments (Becton Dickinson, Foster City, CA), Epics C (Coulter Epics Division, Hialeah, FL), MOFLO (Cytomation, Colorado Springs, Colo.), MOFLO- XDP (Beckman Coulter, Indianapolis, IN). In general, flow cytometry techniques involve the separation of cells or other particles in a liquid sample. Typically the purpose of a flow cytometer is to analyze the isolated particles for one or more properties of them (eg, the presence of a labeled ligand or other molecule). The particles are passed one by one by the sensor and classified based on size, refraction, light scattering, opacity, illuminance, shape, fluorescence, etc.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a flavin-based fluorescent protein variant with improved fluorescence intensity.

(Ii) In addition, the present invention provides a gene encoding a flavin-based fluorescent protein variant.

(Iii) The present invention overcomes the limitations of the detection system using the conventional fluorescent protein in which the fluorescence intensity differs depending on the presence and concentration of oxygen, and the limitations of the fluorescent proteins using the FMN cofactor, which is difficult to use due to the low fluorescence signal. As a technology that can be used, it can be widely used in various biotechnology fields such as life science research, biosensor, biochip, and disease diagnosis.

1 shows a sorting process using KOFP-7 primary and secondary libraries.
Figure 2 shows the enrichment test of the library resulting from the KOFP-7 primary screening results.
3 shows the enrichment test of the library resulting from KOFP-7 secondary screening.
4 shows FACS fluorescence intensities of SY19, SMFP-8, and SMFP-9 finally selected from KOFP-7 primary and secondary libraries.
5 shows the analysis of the emission fluorescence patterns of SB2, KOFP-7 and variants.
Figure 6 shows the quantum yield of SB2, KOFP-7 and variants.
7 shows the results of comparing the sequences of KOFP-7 variants and wild-type SB2 and KOFP-7.
Figure 8 shows the location where the mutations of KOFP-7 variants were introduced.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

<Example 1> KOFP-7 variant library (library) production

The FMN-based fluorescent protein (KOFP-7, SEQ ID NO: 2) derived from Pseudomonas putida was transferred to PEQ-80 vector (Qiagen) using Bam HI, Hin dIII (New England Biolab) restriction enzymes sites, and KOFP-7 The expression plasmid was completed. This plasmid was used as a template, and mutations were introduced into each using the error prone PCR technique. Primers for error prone PCR were 5`-CATCACCATCACCATCACGGATCC-3` and 5`-AAGCTTAATTAGCTGAGCTTGGACTCCTG-3`, and the error rate was adjusted to contain 0.5% error to prepare a library insert. In order to put the prepared library insert into the expression vector, Bam HI and Hin dIII (New England Biolab) restriction enzymes were treated and ligation with the PEQ-80L vector treated with the same restriction enzyme was performed. Then E. coli Jude1 ((F' [Tn 10( Tet ^r ) proAB ⁺ lacI ^q Δ( lacZ )M15] mcrA Δ( mrr - hsdRMS - mcrBC ) f80d lac ZΔM15 Δ lacX74 deoR recA1 araD139 Δ( ara leu )7697 galU galKup ), a giant KOFP-7 variant library was constructed, and a secondary library was constructed using the first discovered variant.

<Example 2> Bacterial culture and KOFP-7 variant library search using flow cytometry

To search for the constructed KOFP-7 variant library, 1 ml of mutant library cells transformed in E. coli Jude1 cells were added to 2% (w/v) glucose and ampicillin (40 μg/mL) in 2% (w/v) glucose at 37°C and 250 rpm shaking conditions. Each was put into the included Terrific Broth (TB) medium and cultured for 4 hours. Shake-cultured library cells were inoculated in TB medium at 1:100 and then cultured until an _{OD 600 reached 0.5 by shaking at 37° C. 250 rpm.} After incubation at 25° C. for 20 minutes for cooling, 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induce expression. After the culture was completed, the cells were recovered, washed twice with PBS, and sorted to recover the top 2% cells showing high fluorescence using a flow cytometry (S3 sortor (Bio-rad)). In order to increase the purity of high cells, the sorted cells were resorted again. The resorted sample was immediately inoculated in TB+2% (w/v) glucose containing ampicillin (40 μg/mL) and cultured overnight. The next day, 1:100 in TB medium containing ampicillin (40 μg/mL). After inoculation, the cells with high fluorescence of the next round were sorted. In this way, sorting and resorting were performed through each library 4 times (Fig. 1). As the round progressed, the sensitivity of the detector was lowered to measure (it can be found that the fluorescence intensity is higher by lowering the sensitivity of the detector of the flow cytometer). In addition, a second library was constructed based on SY19 discovered in the first library in the same manner as above, and sorting and resorting were performed in 4 times (FIG. 1).

<Example 3> Fluorescence-enhanced KOFP-7 variant selection

KOFP-7 In the primary secondary library, it was confirmed whether a group with improved fluorescence intensity was obtained by 4 rounds of sorting and resroting process (Figs. 2 and 3), and KOFP-7 showing high fluorescence by analyzing individual clones in the confirmed group. The variants were identified through a fluorescence signal using a flow cytometer. Among them, three KOFP-7 variants (SY19 (SEQ ID NO: 3), SMFP-8 (SEQ ID NO: 4), SMFP-9 (SEQ ID NO: 5)) were secured higher than the fluorescence intensity of KOFP-7. I could do it (Fig. 4).

<Example 4> Analysis of the emission pattern of the selected KOFP-7 variants

The emission patterns of KOFP-7 variants were investigated. Each purified protein of 3 μM was excited by using a wavelength of 450 nm, and the resulting wavelength was measured. As a result of measuring the emission wavelength between 470 and 600 nm, two peaks were observed at 495 nm and 520 nm. The overall fluorescence pattern of wild-type SB2 (SEQ ID NO: 1) and its mutant were similar, and the emission intensities of SMFP-8 and SMFP-9 were improved by 10 and 11 times, respectively (FIG. 5).

<Example 5> Quantum yield analysis of KOFP-7 variants

Quantum yield is a value representing the ratio of the number of photons or the number of emitted photons to the number of absorbed photons in light emission or photoelectron emission following absorption of light. In other words, the higher the quantum yield, the higher the conversion rate of absorbed light photons into photons that are emitted again. Therefore, a protein with a high quantum yield indicates that it is a bright fluorescent protein. Fluorescent proteins with high quantum yield can be measured to have relatively high fluorescence even when there is a small amount, which leads to an increase in sensitivity. Fluorescein was used as a reference material to measure the quantum yield of the KOFP-7 variant. Compared to the wild-type SB2 with a quantum yield of 0.065, SMFP-8 and SMFP-9 showed improved quantum yields of 0.71 and 0.78, respectively (FIG. 6). Sequences of the discovered variants are shown in FIGS. 7 and 8.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation Kookmin University Industry Academy Cooperation Foundation <120> Flavin mononucleotide binding protein variants derived from Pseudomonas putida with enhanced fluorescence intensity <130> HPC-9527 <150> KR 10-2019-0110449 <151> 2019-09-06 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 142 <212> PRT <213> Pseudomonas putida <400> 1 Met Ile Asn Ala Lys Leu Leu Gln Leu Met Val Glu His Ser Asn Asp 1 5 10 15 Gly Ile Val Val Ala Glu Gln Glu Gly Asn Glu Ser Ile Leu Ile Tyr 20 25 30 Val Asn Pro Ala Phe Glu Arg Leu Thr Gly Tyr Cys Ala Asp Asp Ile 35 40 45 Leu Tyr Gln Asp Cys Arg Phe Leu Gln Gly Glu Asp His Asp Gln Pro 50 55 60 Gly Ile Ala Ile Ile Arg Glu Ala Ile Arg Glu Gly Arg Pro Cys Cys 65 70 75 80 Gln Val Leu Arg Asn Tyr Arg Lys Asp Gly Ser Leu Phe Trp Asn Glu 85 90 95 Leu Ser Ile Thr Pro Val His Asn Glu Ala Asp Gln Leu Thr Tyr Tyr 100 105 110 Ile Gly Ile Gln Arg Asp Val Thr Ala Gln Val Phe Ala Glu Glu Arg 115 120 125 Val Arg Glu Leu Glu Ala Glu Val Ala Glu Leu Arg Arg Gln 130 135 140 <210> 2 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> KOFP-7 <400> 2 Met Ile Asn Ala Lys Leu Leu Gln Leu Met Val Glu Tyr Ser Asn Asp 1 5 10 15 Gly Ile Val Val Ala Glu Pro Glu Gly Asn Glu Ser Ile Leu Ile Tyr 20 25 30 Val Asn Pro Ala Phe Glu Arg Leu Thr Gly Tyr Cys Ala Asp Asp Ile 35 40 45 Leu Tyr Gln Asp Gly Arg Phe Leu Gln Gly Glu Asp His Asp Gln Pro 50 55 60 Gly Ile Ala Ile Ile Arg Glu Ala Ile Arg Glu Gly Arg Pro Cys Cys 65 70 75 80 Gln Val Leu Arg Asn Tyr Arg Lys Asp Gly Ser Leu Phe Trp Asn Glu 85 90 95 Leu Ser Ile Thr Pro Val His Asn Glu Ala Asp Arg Leu Thr Tyr Phe 100 105 110 Ile Gly Thr Gln Arg Asp Val Thr Ala Gln Val Phe Ala Glu Glu Arg 115 120 125 Met Arg Glu Leu Glu Ala Glu Val Ala Glu Leu Arg Arg Gln 130 135 140 <210> 3 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> SY19 <400> 3 Met Ile Asn Ala Lys Leu Leu Gln Leu Met Val Glu Tyr Ser Asn Asp 1 5 10 15 Gly Ile Val Val Ala Lys Pro Glu Gly Asn Glu Ser Ile Leu Ile Tyr 20 25 30 Val Asn Pro Ala Phe Glu Arg Leu Thr Gly Tyr Cys Ala Asp Glu Ile 35 40 45 Leu Tyr Gln Asp Gly Ser Phe Leu Gln Gly Glu Asp His Asp Gln Pro 50 55 60 Gly Ile Ala Ile Ile Arg Glu Ala Ile Arg Glu Gly Arg Pro Cys Cys 65 70 75 80 Gln Val Leu Arg Asn Tyr Arg Lys Asp Gly Ser Leu Phe Trp Asn Glu 85 90 95 Leu Ser Ile Ala Pro Val His Asn Glu Ala Asp Arg Leu Thr Tyr Phe 100 105 110 Ile Gly Thr Gln Arg Asp Val Thr Ala Gln Val Phe Ala Glu Glu Arg 115 120 125 Met Arg Glu Leu Glu Ala Glu Val Ala Glu Leu Arg Arg Gln 130 135 140 <210> 4 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> SMFP-8 <400> 4 Met Ile Asn Ala Lys Leu Leu Gln Leu Met Val Glu Tyr Ser Asn Asp 1 5 10 15 Gly Ile Val Val Ala Lys Pro Glu Gly Asn Glu Ser Ile Leu Ile Tyr 20 25 30 Val Asn Pro Ala Phe Glu Arg Leu Thr Gly Tyr Cys Ala Asp Glu Ile 35 40 45 Leu Tyr Gln Asp Gly Ser Phe Leu Gln Gly Glu Asp His Asp Gln Pro 50 55 60 Gly Ile Ala Ile Ile Arg Glu Ala Ile Arg Glu Gly Arg Pro Cys Cys 65 70 75 80 Gln Val Leu Arg Asn Tyr Arg Lys Asp Gly Ser Leu Phe Trp Asn Glu 85 90 95 Leu Ser Ile Ala Pro Val His Asn Glu Ala Asn Arg Leu Thr Tyr Phe 100 105 110 Ile Gly Thr Gln Arg Asp Val Thr Ala Gln Val Phe Ala Glu Glu Arg 115 120 125 Met Arg Glu Leu Glu Ala Glu Val Ala Glu Leu Arg Arg Gln 130 135 140 <210> 5 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> SMFP-9 <400> 5 Met Ile Asn Ala Lys Leu Leu Gln Leu Met Val Glu Tyr Ser Asn Asp 1 5 10 15 Gly Ile Val Val Ala Lys Pro Glu Gly Asn Glu Ser Ile Leu Ile Tyr 20 25 30 Val Asn Pro Ala Phe Glu Arg Leu Thr Gly Tyr Cys Ala Asp Glu Ile 35 40 45 Leu Tyr Gln Asp Gly Ser Phe Leu Gln Gly Glu Asp His Asp Gln Pro 50 55 60 Gly Ile Ala Ile Ile Arg Glu Ala Ile Arg Glu Gly Arg Pro Cys Cys 65 70 75 80 Gln Val Ile Arg Asn Tyr Arg Lys Asp Gly Ser Leu Phe Trp Asn Glu 85 90 95 Leu Ser Ile Ala Pro Val His Asn Glu Ala Asp Arg Leu Thr Tyr Phe 100 105 110 Ile Gly Thr Gln Arg Asp Val Thr Ala Gln Val Phe Ala Glu Glu Arg 115 120 125 Met Arg Glu Leu Glu Ala Glu Val Ala Glu Leu Arg Arg Gln 130 135 140

Claims (15)

As an FbFP variant with increased fluorescence intensity compared to flavin-based fluorescent protein (FbFP), the FbFP variant contains a part of the amino acid sequence of FbFP in SEQ ID NO: 1, H (Histidine), which is the 13th amino acid in the amino acid sequence of FbFP, is Y (Tyrosine); The 23rd amino acid, Q (Glutamine), is P (Proline), and the 53rd amino acid, C (Cysteine), is G (Glycine); 108th amino acid Q (Glutamine) to R (Arginine); 112th amino acid Y (Tyrosine) to F (Phenylalanine); 115th amino acid I (Isoleucine) to T (Threonine); And an FbFP variant in which the 129th amino acid, V (Valine), is substituted with M (Methionine).
The FbFP variant according to claim 1, wherein the FbFP and FbFP variants are derived from Pseudomonas putida.
According to claim 1, wherein the FbFP variant is E (Glutamate), which is the 22nd amino acid of the amino acid sequence of FbFP of SEQ ID NO: 1 is K (Lysine); The 47th amino acid, D (Aspartate), is E (Glutamate); 54th amino acid R (Arginine) to S (Serine); And FbFP variant further comprising that the 100th amino acid T (Threonine) is substituted with A (Alanine).
The FbFP variant according to claim 3, wherein the FbFP variant further comprises a substitution of N (Asparagine) for D (Aspartate), which is the 107th amino acid in the amino acid sequence of FbFP in SEQ ID NO: 1.
The FbFP variant according to claim 3, wherein the FbFP variant further comprises that L (Leucine), which is the 86th amino acid in the amino acid sequence of FbFP of SEQ ID NO: 1, is substituted with I (Isoleucine).
The FbFP variant according to claim 1, wherein the FbFP variant comprises an amino acid sequence selected from the group consisting of the second sequence, the third sequence, the fourth sequence, and the fifth sequence.
A nucleic acid molecule encoding the FbFP variant of claim 1.
A vector comprising the nucleic acid molecule of claim 7.
The vector according to claim 8, wherein the vector comprises the nucleic acid molecule as a reporter gene, and further comprises a gene encoding a protein of interest to be expressed.
A host cell comprising the vector of claim 8.
A composition for fluorescent coloring comprising the FbFP variant of claim 1, the nucleic acid molecule of claim 7 or the vector of claim 8.
A method for analyzing the expression of a protein of interest comprising the step of expressing the vector of claim 9.
Expressing the vector of claim 9 to produce a protein of interest; And separating the generated target protein.
A method for preparing an FbFP variant comprising the following steps:
a) culturing a host cell comprising a vector containing a nucleic acid molecule encoding the FbFP variant of claim 1; And
b) recovering the FbFP variant expressed by the host cell.
A method for screening FbFP variants comprising the following steps:
a) constructing a library of the FbFP variant of claim 1 or a nucleic acid molecule encoding the FbFP variant or a nucleic acid molecule encoding the FbFP variant to which a random point mutation is added to the nucleic acid molecule encoding the same; And
b) selecting an FbFP variant or a nucleic acid molecule encoding the FbFP variant with increased fluorescence intensity than that of the FbFP protein of SEQ ID NO: 1 in the library.

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