KR20100119104A - Fast maturating red fluorescent protein, fmred, as a novel reporter and molecular probe - Google Patents

Abstract

PURPOSE: A red fluorescent protein FmRed(fast maturating red fluorescent protein) is provided to use in continuous reaction system such as fluorescence resonance energy transfer(FRET). CONSTITUTION: A red fluorescent protein FmRed is formed by elongation of MGS amino acids at N-terminal and CGSPG amino acids at C-terminal. The FmRed has an amino acid sequence of sequence number 2. A FmRed gene contains nucleotide sequence of sequence number 1. A method for preparing FmRed using elongation mutagenesis PCR comprises: a step of performing PCR of defective gene of wild type DsRed to construct a mutant gene library; a step of introducing the library into each cell to express mutant protein; and a step of selecting mutant protein having quick fluorescence and dark red.

Description

Fast maturating red fluorescent protein, FmRed, as a novel reporter and molecular probe, as a new reporter and molecular probe

The present invention relates to a red fluorescent protein inducing fast and strong fluorescence as a new reporter and a molecular probe, and more particularly to the three amino acids MGS and C-terminal at the N-terminus of the wild type Red Fluorescence Protein (DsRed) gene. The red fluorescent protein FmRed (fast maturating red fluorescent protein) consisting of five amino acids CGSPG is extended.

Fluorescent proteins are proteins that fluoresce by emitting light of a specific wavelength in the process of absorbing and activating when receiving light of a certain wavelength and then returning to a normal state again. The fluorescent protein is particularly important in biotechnology research, and can be easily identified by the expression process or intracellular location of the target protein by binding to the N- or C-terminus of the protein to be characterized. In addition, it is generally used in the process of determining the stability or expression in the cell, and from the medical point of view it is also used to locate cancer cells or tumors.

As in the case of using the fluorescent protein, there is a method of easily identifying the intra- and physiological-ecological changes in the cell, and the method of relying on enzyme activity after injecting a chemical or precursor such as a fluorescent pigment into the cell. . The method can easily and accurately observe physiological changes, but poses many risks due to the toxicity of the injected chemicals or precursors, ie mutating or inducing apoptosis during DNA replication, for example. have.

However, since fluorescent proteins fluoresce themselves without substrates or cofactors, they have very few effects on metabolites or interference with cytophysiological factors. Therefore, even in the state of living cells that maintain normal physiology, it is possible to observe the movement of specific proteins, active pathways, cell division and differentiation. These characteristics make it a better reporter or probe than any existing method for monitoring physiological phenomena in vivo without time lag. This is because it is a breakthrough technology that can directly observe the complex changes occurring inside the protein without destroying cells or organisms through fluorescence caused by light of a specific wavelength inherent under various probe conditions.

Typical fluorescent proteins currently used include green fluorescence protein (GFP), which represents green fluorescence, red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue blue fluorescent protein (BFP), and yellow Many types of fluorescent proteins, such as YFP (yellow fluorescent protein), have been developed or improved and are used for research and commercial purposes.

The fluorescence induction process of the fluorescent protein currently being developed and used is so diverse that almost all wavelengths from ultraviolet to visible light can be used here. Among them, the interest in red fluorescent protein (DsRed) using a wavelength near infrared light, which can be directly observed by the naked eye and has low cytotoxicity, is gradually increasing.

The red fluorescent protein (DsRed) has a structure similar to the fluorescent proteins known in the art to exhibit red fluorescence derived from coral. Fluorescent proteins generally have a solid tertiary structure with fluorophores inside the cylindrical structure of the folded β-sheet. However, in the case of red fluorescent protein (DsRed), four such cylindrical structures are gathered and assembled into one protein to show red fluorescence. Because of the above characteristics, it is very stable and has a high wavelength excitation and emission value compared to other fluorescent proteins, so it can be easily identified with the naked eye. Therefore, there is an advantage that the damage caused by the excitation wavelength of the other fluorescent protein mainly depends on the ultraviolet rays of the low region, that is, the damage of nucleic acids, fatty acids, or amino acid residues, which are the main molecules in vivo, can be minimized.

In addition, since it has a relatively strong resistance to photobleaching phenomenon, the color development time is long, reproducible in repeated experiments, and excellent in pH conditions, it can fluoresce in an environment of pH 5-12. Therefore, it is known to be very stable physically and chemically and to have strong resistance to denaturing agents and organic solvents. Because of these advantages, the use of red fluorescent protein (DsRed) is preferred, but since four monomers gather together to form one protein, it takes longer maturation time to form an active (quaternary) structure than other fluorescent proteins. Has Many variants have been reported to exemplify the slowness of the quaternary structure formation and active structure formation time as illustrated in Table 1 below.

In particular, for the rapid expression of red fluorescent protein (DsRed), similar to other fluorescent proteins, results have been reported for the fluorescence variant using only monomer, but the quantum yield (low quantum yield), which was the advantage of wild type DsRed It has been analyzed that there is a problem that stable characteristics are lost in photobleaching phenomenon.

Most of the mutant proteins that have been induced to transform wild-type DsRed into templates are involved in the formation of fluorophores, or by replacing some of the surrounding amino acids, which is assumed to be the prototype of all fluoroproteins (Green Fluorescent Protein, GFP) is a method that uses elongation as an arbitrary extension to compensate for amino acid deficiency of shortened terminal during evolution.

The mutated proteins obtained by the above method have a slightly different structure of the fluorescent terminal that receives light, absorbing and emitting light of different wavelengths, and exhibiting fluorescence of different colors, but essentially have red wavelengths. However, the introduction of the same dimensions (amino acid length-like sequences) as the Green Fluorescent Protein (GFP) for the induction into the monomer results in the loss of structural stability of the quaternary structure, especially the resistance to photobleaching. And causing a decrease in quantum yield.

Therefore, in the present invention, a novel red fluorescent mutant protein which has matured without destroying the structural characteristics of DsRed, that is, quaternary structure, and has excellent resistance to quantum yield and photobleaching, was prepared and confirmed its effect, thereby completing the present invention.

An object of the present invention is to provide a novel red fluorescent mutant protein and a gene of the new red fluorescence mutant protein, which have excellent resistance to quantum yield and photobleaching due to the structural stability of the quaternary structure.

It is also an object of the present invention to provide a recombinant expression vector comprising the new red fluorescent protein gene and a transformed microorganism transformed with the recombinant expression vector.

It is also an object of the present invention to provide a fusion protein comprising the novel red fluorescent mutant protein as a reporter or fusion tag.

It is also an object of the present invention to provide a biosensor and operon trap vector comprising the new red fluorescent protein gene as a reporter or fusion tag.

In order to achieve the above object, the present invention provides a red fluorescent protein FmRed (fast maturating red) in which three amino acids MGS at the N-terminus of the wild type DsRed (Red Fluorescence Protein) gene and CGSPG which are five amino acids at the C-terminus are extended. fluorescent protein).

The present invention provides a red fluorescent protein FmRed (fast maturating red fluorescent protein) consisting of the amino acid sequence of SEQ ID NO: 2, the red fluorescent protein consisting of the nucleotide sequence of SEQ ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 2 FmRed (fast maturating red fluorescent protein) gene.

The present invention

a) for a defective gene of wild type DsRed, performing a polymerase chain reaction with a random combination primer added with 3 to 10 amino acids in the defective gene to construct a variant gene library;

b) introducing each of the constructed libraries into cells to select and express individual variant proteins; And

c) selecting the desired variant protein from the expressed variant protein, which exhibits faster fluorescence and darker red color than the wild type DsRed; It provides a method of producing a red fluorescent protein FmRed (fast maturating red fluorescent protein) using an elongation mutagenesis polymerase chain reaction, including.

Hereinafter, the configuration of the present invention with reference to the accompanying drawings will be described in detail.

DsRed used as a template of the present invention is a marine-derived protein that exhibits red fluorescence and forms a tetramer structure of four monomers of about 28 KDa. As a fundamental problem of the DsRed, in order to improve the slow expression time and to compensate for the shortcomings of the DsRed variants (mainly monomers) which exhibit relatively fast fluorescence but have low quantum yield and are sensitive to photobleaching, the quaternary structure is basically maintained but fast. In order to induce the maturation process and high fluorescence rate, a mutation library designed by randomly increasing both ends is used.

More specifically, the mutant library includes a start codon (ATG) and a stop codon (TAG) at both ends of the wild-type DsRed gene, and a wild type DsRed defective gene in which the base following the start codon is frame shifted. , Mutant gene libraries were prepared by polymerase chain reaction with primers added with 3 amino acids and 10 amino acids, see FIG. 2.

The primers added with 3 to 10 amino acids include a start codon (ATG) and a stop codon (TAA) at both ends of the defective gene of the wild-type DsRed, each having a base sequence substituted with NNN. Polymerase using (N-terminal primer, 5'-ATG NNN (NNN) n A GCC TCC TCC G-3 '; C-terminal primer, 5'-TAA (NNN) n CAG GAA CAG G-3') A chain reaction was performed.

After introducing the constructed libraries into cells, the individual mutant proteins were selected and expressed, and then the mutant protein expressed in the expressed mutant protein was 10 to 12 hours faster than the wild type DsRed. Selected.

More specifically, in order to compare the characteristic difference between the selected mutated protein and wild-type DsRed, it was activated in a continuous operation in an LB solid medium and cultured under the same conditions and analyzed for expression level using a naked eye and a hand UV lamp. While the protein fluoresces around 12 hours, wild-type DsRed has been shown to exhibit non-uniform variation and fluorescence after at least 20 to 24 hours. See FIG. 3.

In addition, the fluorescence was analyzed by culturing each sample in LB liquid medium, and the fluorescence was analyzed. However, although the expression levels of wild-type DsRed and the mutant protein were the same, the selected mutant protein showed superior fluorescence than wild-type DsRed. Confirmed. In other words, in the native-gel loaded with the same amount, the red protein band was clearly observed, but the wild type DsRed showed unclear band morphology not only by the naked eye but also by using a UV lamp. In addition, it was confirmed that the quantum yield was greatly improved. This was again incubated by time zone, and when the protein expression was induced by IPTG and when it was not, the time of maturation was measured by flow cytometry. As a result of the solid medium, the improved maturation process of the mutant protein was confirmed. It became.

Therefore, in the case of the mutant protein according to the present invention, the mutant effect is accelerated in the maturation process rather than the increased expression rate and shows rapid fluorescence. It can be verified. See FIG. 4.

The protein expressed in the clone showing the rapid maturation process was named as the red fluorescent protein FmRed (fast maturating red fluorescence protein), and then 3 and 5 amino acids at the N-terminus and C-terminus, respectively, through full sequencing. It was confirmed that this added variant. More specifically, the selected mutated protein according to the present invention is a red fluorescent protein FmRed (fast) formed by extending three amino acids MGS at the N-terminus of the wild type DsRed (Red Fluorescence Protein) gene and five amino acids CGSPG at the C-terminus. maturating red fluorescent protein). Please refer to Fig.

Provided is a recombinant expression vector comprising a red fluorescent protein FmRed gene according to the present invention and a transformed microorganism which is deposited with accession number KCTC11464BP, which is transformed with the recombinant expression vector.

More specifically, the red fluorescence protein FmRed selected according to the present invention is reintroduced back into the pTrc99A plasmid, thereby recombining the recombinant plasmid (pFmRed), which maintains excellent characteristics and activity as a reference for selection, to E. coli XL1-Blue. The transformed strain ( E. coli XL1-Blue [pFmRed]) was deposited on February 10, 2009 with Genebank, an international depository institution located at the Biotechnology Research Institute, Eue-dong, Yuseong-gu, Daejeon, Korea. KCTC11464BP was given.

The present invention provides a fusion protein comprising the red fluorescent protein FmRed gene according to the present invention as a reporter or a fusion tag.

More specifically, the wild type DsRed and the mutant protein according to the present invention were tested by mounting the reporter behind a fusion tag or an active promoter under the same conditions, and as a result, the characteristics of the DsRed fusion tag were observed in the mutant protein FmRed. It was confirmed that the probe can be detected within 10 to 12 hours at a time and a cell amount that cannot be observed in the wild type DsRed as a reporter.

The present invention also provides a biosensor and operon trap vector comprising the red fluorescent protein FmRed gene according to the invention as a reporter or fusion tag.

More specifically, in the case of the pBGR1 promoter trap vector, which is an operon trap vector according to the present invention, two fluoroprotein reporters are positioned to face each other, and a promoter trapped in the reporter direction of the green fluorescence protein or Function expression by regulators can be monitored within about 10 hours, and the regulator or promoter that has been trapped on the other side can be identified by attaching FmRed as a reporter. Fluorescence was confirmed to the extent that it can be confirmed by naked eyes. As a result, it was finally confirmed that the new mutant protein is actually applicable to fluorescence protein linkage or continuous reaction system such as dual reporter system or fluorescence resonance energy transfer (FRET).

The red fluorescent protein FmRed according to the present invention has a stronger fluorescence in a faster time than wild type DsRed, and due to the characteristics of the existing wild type DsRed, as well as new sensitivity and rapid characteristics, gene cloning, promoter trap, fusion protein production, In addition to measuring intracellular protein transport, protein interactions and the presence of oxygen, it is widely used in a variety of biosensing applications including yeast two hybrids and fluorescence resonance energy transfer systems.

The red fluorescent protein FmRed according to the present invention not only compensates for the fatal shortcomings of DsRed, but has also been supplemented to the shortcomings of the reported variants, and has been applied to various cell physiology, immunity, engineering, and medicine fields for higher efficiency and reliability. Can be obtained quickly, contributing to biotechnology-life science research.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.

At this time, if there is no other definition in the technical and scientific terms used, it has a meaning generally understood by those of ordinary skill in the art.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

[ Example  1] Mutation Library Construction and Clonal Screening with Fast Fluorescence

The construction of mutation libraries for the selection of fast fluorescence-induced high sensitivity mutagenesis was performed by elongation mutagenesis polymerase extension using custom primers. To this end, wild-type DsRed (Red Fluorescence Protein) gene was first amplified by polymerase extension from pDsRed-N1 (Clontech) and cloned into Nco I and Hind III sites of pTrc99A (pharmacia) plasmid. After confirming the structure of the cloned complete gene and the fluorescence of the expressed protein, the method of selecting the mutant strain of the restored function to easily determine whether the kidney mutation is induced (Protein Eng. 2001, 14: 647-654) Based functional defeats were induced by base removal of the distal sites. This causes frame shift by removing the start codon (ATG) and the end codon (TAG) at both ends while recloning the DsRed gene into the pTrc99A vector, and additionally missing the base following the start codon. Could be implemented. In the case of the produced defective gene, after confirming that the function of the red fluorescent protein was lost, the sequence was analyzed and verified.

Synthetic primers (N-terminal primers, 5'-ATG (NNN) n AGCCTCCTCCG-3) to restore the normal open reading frame (ORF) at the same time as the kidney is induced in the resulting defective gene template C-terminal primer, 5'-TAA (NNN) n CAGGAACAGG-3 ') was introduced to perform the polymerase extension reaction and then the library was prepared by subcloning (Fig. 2).

In the case of the prepared mutant library protein, 3 to 10 peptides were extended at both ends, and artificially induced frame shifts were corrected, and thus, kidney mutations could be easily induced by selecting fluorescent clones. After transforming the recombinant plasmid containing the mutated gene into Escherichia coli XL1-Blue, the prepared library was incubated for 24 hours in an LB solid medium, and the primary mutated enzymes with restored function were selected (about 4000), compared to wild-type DsRed. Clones showing rapid fluorescence and showing deep red were selected as final candidates. Among them, clones showing the fastest expression time of about 10 to 12 hours and showing bright fluorescence compared to DsRed were finally selected.

After separating the plasmids of the selected clones, it was confirmed that the same results were obtained by repeating the process of separating the included genes and re-introducing them into another plasmid.

The final selected clones were cultured in LB solid medium and liquid medium containing 50 ul / ml of ampicillin in a 37 ° C. incubator and measured for fluorescence time, as can be seen from the results in FIG. 3. Wild-type DsRed showed fluorescence when cultured for 24 hours, whereas the final selected mutant protein showed vivid red fluorescence in cultured colonies for at least 12 hours and up to 15 hours. As a result of analyzing the gene sequences of the selected clones, as can be seen from the nucleotide sequence and amino acid sequence of FIG. 1, three and five amino acids are extended at both ends of the DsRed sequence, that is, the N-terminal and the C-terminal, respectively. Confirmed variants. This was named FmRed (fast maturating red fluorescence protein) and the characterization experiment was conducted.

[ Example  2] with interfering substances Metabolites  Present In crude extract  Fluorescence

The characteristics of FmRed, the mutant protein selected in Example 1, are generally characterized by the ability to operate fluorescent proteins as probes, ie, in crude extracts containing interferences, metabolites, and various proteolytic enzymes. Compared to wild type DsRed. For comparison under the same conditions, pTrc-DsRed and pTrc-FmRed were constructed using Nco I and Hind III restriction enzymes in the pTrc-99A plasmid only for the full open reading frame (ORF) of wild type DsRed and the mutant protein FmRed. It was.

Comparing DsRed and FmRed on solid medium under various culture conditions, it was confirmed that DsRed exhibited a deviation of expression until fluorescence was detected and that 1.5 to 2 times more time was required than FmRed. When IPTG was added as a protein expression inducing agent to the solid medium, the difference in expression induction time was reduced, but it was confirmed that the difference of about 3 hours was still present.

The results were similarly observed in the general detection method of red fluorescent protein, i.e., when using the naked eye or UV lamp. In order to confirm the change of protein and structural characteristics in the actual solution, the crude enzyme solution was prepared and analyzed by SDS-PAGE and native gel (Native-gel), and then experiments were performed to compare the fluorescence characteristics on the gel.

The cells used in the experiment were used to induce protein expression by IPTG or incubated at 37 ° C. for 30 hours without adding an expression inducing agent. No significant difference was observed when the amount of expression of DsRed and FmRed in the whole protein was compared by SDS-PAGE in the whole cell state before cell disruption. Therefore, it could be verified that the result of increased fluorescence or rapid maturation time was not due to the difference in expression level of the mutant protein.

To investigate the fluorescence characteristics of crude extract, crude enzyme solution was prepared according to the following procedure.

Cells cultured without the expression inducing agent were diluted or concentrated to have an absorbance value of 2.0 and washed three times with 300 mM of 50 mM Tris-HCl. The cells were pulverized by sonication with pulses at 10 second intervals and left for 50 seconds, and then precipitated for 10 minutes using a 13,000 rpm 4 ° C centrifuge. Only the supernatant of the centrifuged sample was recovered, the total amount of protein was measured, and each sample was calibrated to have the same absorbance and diluted with 50 mM Tris-HCl solution. 14 ul of each sample, 4 ul of 5X SDS loading dye, and 2 ul of 1M DTT were mixed and boiled at 100 ° C. for 10 minutes to induce protein denaturation, followed by electrophoresis at 100 V voltage on SDS-PAGE for 2 hours.

After the loaded sample was completely separated on the gel, the PAGE was separated, stained for 4 hours, and bleached by soaking in a solution containing 30% methanol and 10% acetic acid.

As a result, the consensus band of FmRed was confirmed near 28 KDa, the size of wild type DsRed.

In order to compare the fluorescence intensity of stably expressed FmRed with DsRed under the same conditions, 16 ul of each sample and 5X native loading dye were mixed, followed by electrophoresis for 2 hours at a voltage of 100 V on an unmodified gel without SDS. It was. In order to confirm the degree of development, the location of the loaded dye was checked and developed by adjusting the electrophoresis time, and then the gel was separated and washed with distilled water. The washed gel was irradiated with a hand UV lamp to confirm the position of the protein band. As expected, the band of FmRed could be confirmed at the same position as tetramer DsRed, and it was confirmed that FmRed showed brighter and brighter fluorescence than DsRed even though it contained the same amount of protein. Therefore, the mutant protein FmRed has a wild-type structure and the quantum yield related to the intensity of fluorescence is improved.

[ Example  3] FACS Using DsRed Wow FmRed In vivo expression time

In order to more accurately identify the protein expression time in the clone, a single colony was inoculated in LB solid medium containing 50 ul / ml of ampicillin and incubated at 37 ° C., and the fluorescence expression levels of DsRed and FmRed were confirmed. As can be seen from the results of Figure 3, in the case of DsRed 21 hours after the fluorescence can be confirmed in a single colony, FmRed was able to confirm the fluorescence in a single colony before 15 hours. Therefore, it was confirmed that the expression time of FmRed is about 7 hours faster in vivo than DsRed.

DsRed and FmRed were inoculated in LB liquid medium containing 50 ul / ml of ampicillin under the same conditions as in the solid medium, and the samples were taken at intervals of 2 hours while shaking culture at 37 ° C. and 200 rpm, and loaded onto native-gel. By comparing the time the fluorescence appeared. The cell amounts of each sample recovered were equally corrected and then crushed under mild conditions to electrophores the same amount of protein.

As a result, in the case of DsRed, the fluorescent protein could not be identified after 8 hours from the start of the incubation time, but in the case of FmRed, the fluorescence could be confirmed from 2 hours, and the degree of fluorescence rapidly increased over time. Confirmed.

In order to obtain more accurate results, the fluorescence of the recovered cells was measured by time after inoculating the minimum cells using a Fluorescence Activated Cell Sorter (FACS). As a result, as can be seen in Figure 4A, FmRed did not induce protein expression was confirmed that the fluorescence appeared from the culture time of 20 hours. On the other hand, DsRed did not show a clear fluorescent clone even after 42 hours of incubation under the same conditions.

In addition, when inducing expression at the basic level without addition of IPTG, it is difficult to confirm fluorescent protein expression of wild-type DsRed unlike FmRed, which shows little variation depending on cell physiology or culture conditions. The characteristics were compared. Expression of the protein was induced in LB liquid medium containing 50 ul / ml of ampicillin and induced by adding 1 mM of IPTG when the cell absorbance reached 0.4. After that, samples were shaken at 37 ° C. and 200 rpm, and sampled at 1 hour intervals, and the time of fluorescence expression using FACS was compared. As a result, as can be seen in Figure 4 B, it was confirmed that the fluorescence appeared from the time when the culture time of DsRed is 8 hours, the time of FmRed is 7 hours. Overexpression of the protein overexpressed the variation of expression time, but the fluorescence peak of FmRed was pushed backward compared to that of DsRed. In the above results, the fluorescence expression time during cell culture was varied depending on the inoculated cells, inoculation amount, and whether protein expression was induced. However, as a general result, the characteristics of FmRed with fast maturation time and excellent fluorescence are obvious. It was confirmed to be maintained.

[ Example  4] Affinity Chromatograph  Used DsRed Wow FmRed  Separation tablet

In order to confirm the change in the properties and functions of the protein by any amino acid residues of the FmRed introduced in Example 1 and to determine the exact structure, fluorescence intensity, light stability and the like was purified purification. The most common DsRed purification method known is a method of separating a protein expressed with a His-tag at the N- or C-terminus into a Ni-NTA column. However, if the his-tag is linked to DsRed, it affects the protein's natural structure, making it difficult to identify functionally intact protein activity. Moreover, in the case of the mutant protein FmRed, the protein is extended at both ends, and their effects may be canceled by the tag attached additionally or may have other effects. Therefore, it is very important to selectively separate only intact proteins.

In addition, wild type DsRed, which is a prototype of the mutant protein FmRed, is known to be capable of weak ionic bonds with copper, and is used as a copper-biomarker capable of measuring copper in vivo or in samples. This characteristic is known because eight histidines and one cysteine in the protein structure can bind to copper, and most histidines and cysteines are made by combining four monomers. It is known to be possible because it is exposed to the quaternary structure surface.

In the case of the FmRed of the present invention, since the basic amino acid sequence is the same as that of DsRed and one cysteine is additionally inserted at the N-terminus of the newly extended amino acid sequence, the basic structural characteristics of the FmRed are expected to be similar. Based on this, separation and purification were attempted using a metal affinity column with copper as in the case of DsRed.

First, the bare resin (chelating sepharose, GE Healthcare) without metal ions was washed with sterile tertiary distilled water, and then 0.5 ml of 0.1 mM copper sulfate (CuSO ₄ ) was flowed into the resin. Attached. Thereafter, unbound copper and salt components were removed using 5 ml of weakly acidic buffer having the composition shown in Table 2 below. After 5 ml of binding buffer was flowed to the prepared column, the equilibrium was reached, and the FmRed coenzyme solution mixed with the same buffer was loaded to induce protein adhesion. 10 ml of Wash Buffer I was flowed. Next, additional 10 ml of Wash Buffer II was added to remove the non-binding proteins and nonspecific weakly bound impurities and proteins, followed by selective binding by addition of an elution buffer containing 4 mM immidazole. Recovered protein was recovered. The flow rate of all processes was fixed at 1 ml / min, and the wild type DsRed protein was also purified by the same process. As a result of analysis after purification, as shown in FIG. 5, a protein having more than 90% purity could be separated by a single process.

[Example 5] Comparison of protein properties of purely isolated DsRed and FmRed

(1) Comparison of quantum yield between DsRed and FmRed

In general, wild type DsRed has a maximum excitation wavelength at 558 nm and an emission wavelength at 583 nm. FmRed showed brighter fluorescence than wild-type DsRed, and was scanned using a fluorometer (LS55) to determine whether it had different excitation and emission wavelengths.

As a result, as can be seen in Figure 6, it was confirmed that the FmRed has a spectrum and the maximum fluorescence wavelength of the same pattern as DsRed, showing a fluorescence value of 1.7-2 times at the same protein concentration improved the quantum yield is high. Confirmed. Therefore, it was confirmed that FmRed isolated in Example 1 is a mutant protein that maintains the quaternary structure important for photobleaching and accelerates fluorescence and maturation.

(2) Storage stability of FmRed

In general, most proteins in coenzyme fluids that are exposed to solutions or solvents due to cell membrane breakdown are degraded by protease, and have inherent half-life by quality control, and thus lose their natural function. In the course of this experiment, it was confirmed that the fluorescence-induced whole cell enzyme retained its original color for more than one month on solid media, and in the case of the protein in the crude enzyme solution, three weeks passed at 4 ° C. Afterwards, it was confirmed that most of the initial fluorescence was maintained.

(3) Comparison of photobleaching phenomena of DsRed and FmRed

Fluorescent proteins as reporters and probes have a photobleaching effect that reduces initial fluorescence in repeated experiments or when irradiated with a constant wavelength of a certain wavelength for a long time, especially in the case of DsRed variants (see Table 1) made of monomers or dimers. It is known that there is a problem that the fluorescence decreases rapidly over time because most of the excellent light safety of DsRed is lost. In case of FmRed isolated in Example 1, the quaternary structure, which is an important factor for photosafety of the wild-type protein, is maintained, and thus no photobleaching phenomenon occurs, and when the same amount of protein is irradiated with light of 540 to 560 nm, It was finally confirmed to show improved photosafety compared to wild type protein.

Example 6 Functional Evaluation of FmRed as a Fusion Tag and Reporter

(1) Functionality as a fusion tag

Fluorescent proteins are often expressed in the form of folds, localizations, and interaction reporters, fused with other proteins to be traced or characterized. Therefore, when FmRed is expressed in the state of fusion with other proteins, fusion with various kinds of proteins was attempted to confirm whether the FmRed has the fusion ability and stability necessary for the function as a reporter.

As a result, when the plasmid pMAL-c2X was cloned after processing the restriction enzymes of EcoR I and Hind III as shown in FIG. 7A, the fusion protein was soluble in the PAGE results. In B of FIG. 7, the same amount of DsRed fusion protein as in the fused state shows a similar or slightly stronger fluorescence intensity, and in most cases, the same or superior fusion ability as the wild type DsRed was confirmed. Therefore, in the case of FmRed, it was confirmed that there is no problem in the function of the reporter and the probe required in the environment fused with other proteins.

(2) expression factor traps and use as operational reporters

In general, DsRed has a disadvantage in that it is difficult to be used in the same reporter system because it has a significantly different fluorescence expression time compared to other fluorescent proteins (GFP or YFP) because of the time required to make the quaternary structure required for the function. In other words, if the Green Fluorescent Protein (GFP) is monitored in real time simultaneously due to the characteristics of DsRed, which is relatively longer than the aging time in vivo, the expression time of slow DsRed determines the overall monitoring time. In fact, in the case of the pBGR1 promoter trap vector designed by the present inventors, two fluoroprotein reporters are positioned to face each other, and the expression of the function by promoters or regulators trapped in the reporter direction of the green fluorescent protein is 10. It can be monitored within hours, but regulators with functions trapped on the other side require more than 24 hours before the reporter DsRed is expressed and monitored. In other words, when the number of strains in the library or the colonies were small, when the function of the trapped factor was weak, it could be confirmed whether or not it was a reporter after 36 to 48 hours or more. The new FmRed was attached to these trap vector systems to confirm the operation, and as a result, the dual reporter system emits fluorescence that can be seen with the naked eye at the same time as the green fluorescent protein within 10 hours. In addition, it was finally confirmed that it is a novel mutant protein that can be actually applied to fluorescence protein linkage or continuous reaction system such as fluorescence resonance energy transfer (FRET).

Figure 1 shows the nucleotide sequence (A) and amino acid sequence (B) of the red fluorescent protein FmRed according to the present invention,

2 is a schematic of a random terminal extension method for inducing a renal mutation for cloning the red fluorescent protein FmRed according to the present invention,

(A: DsRed gene induction of defects, B: function restored library)

Figure 3 is a result of confirming the fluorescence time of the red fluorescent protein FmRed clone according to the present invention in a solid medium,

4 is a result of confirming the expression time of the red fluorescence of the red fluorescent protein FmRed according to the present invention using a flow cytometer,

(A: when comparing without expression inducer, B: comparing after adding IPTG)

5 is a result of confirming the red fluorescent protein FmRed according to the present invention purified after separation in SDS-PAGE (A) and native-gel (B),

(1: pTrc-DsRed, 2: pTrc-FmRed)

6 is a result of confirming the quantum yield of the red fluorescent protein FmRed according to the present invention,

(A: excitation wavelength, B: emission wavelength)

7 shows the results of fusion stability of red fluorescent protein FmRed according to the present invention in SDS-PAGE (A) and native gel (B).

(A; 1: size marker, 2: pMal-c2X, 3: MBP-FmRed, 4: MBP-DsRed, B; 1: pMal-c2X, 2: MBP-FmRed, 3: MBP-DsRed)

<110> Industry Foundation of Chonnam National University <120> Fast maturating red fluorescent protein, FmRed, as a novel          reporter and molecular probe <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 702 <212> DNA <213> Unknown <220> <223> FmRed <220> <221> gene (222) (1) .. (702) <223> FmRed sequence <400> 1 atgggctcca cagcatcatc cgagaacgtc atcaccgagt tcatgcgctt caaggtgcgc 60 atggagggca ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 120 tacgagggcc acaacaccgt gaagctgaag gtgaccaagg gcggccccct gcccttcgcc 180 tgggacatcc tgtcccccca gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc 240 gacatccccg actacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 300 aacttcgagg acggcggcgt ggcgaccgtg acccaggact cctccctgca ggacggctgc 360 ttcatctaca aggtgaagtt catcggcgtg aacttcccct ccgacggccc cgtgatgcag 420 aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 480 aagggcgaga cccacaaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 540 aagtccatct acatggccaa gaagcccgtg cagctgcccg gctactacta cgtggacgcc 600 aagctggaca tcacctccca caacgaggac tacaccatcg tggagcagta cgagcgcacc 660 gagggccgcc accacctgtt cctgtgtgga tcccccgggt aa 702 <210> 2 <211> 233 <212> PRT <213> Unknown <220> <223> FmRed <400> 2 Met Gly Ser Thr Ala Ser Ser Glu Asn Val Ile Thr Glu Phe Met Arg   1 5 10 15 Phe Lys Val Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile              20 25 30 Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly His Asn Thr Val Lys          35 40 45 Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu      50 55 60 Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala  65 70 75 80 Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp                  85 90 95 Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Ala Thr Val Thr Gln             100 105 110 Asp Ser Ser Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile         115 120 125 Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met     130 135 140 Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu 145 150 155 160 Lys Gly Glu Thr His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr                 165 170 175 Leu Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu             180 185 190 Pro Gly Tyr Tyr Tyr Val Asp Ala Lys Leu Asp Ile Thr Ser His Asn         195 200 205 Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His     210 215 220 His Leu Phe Leu Cys Gly Ser Pro Gly 225 230  

Claims (11)

FmRed (fast maturating red fluorescent protein), wherein the red fluorescent protein (FmRed) is a three-amino acid MGS at the N-terminus of the wild type DsRed (Red Fluorescence Protein) gene and a CGSPG of five amino acids at the C-terminus. The method of claim 1, The red fluorescent protein FmRed is a fast fluorescent red fluorescent protein (FmRed) consisting of the amino acid sequence of SEQ ID NO: 2. A red fluorescent protein FmRed (fast maturating red fluorescent protein) gene, encoding the amino acid sequence set forth in SEQ ID NO: 2. The method of claim 3, wherein A red fluorescent protein FmRed (fast maturating red fluorescent protein) gene consisting of the nucleotide sequence of SEQ ID NO: 1. Recombinant expression vector comprising a red fluorescent protein FmRed gene according to claim 3 or 4. A transformed microorganism transformed with the recombinant expression vector according to claim 5. The method of claim 6, The transforming microorganism will be deposited with the accession number KCTC11464BP. a) constructing a mutant gene library by performing a polymerase chain reaction on a defective gene of wild type DsRed with a primer added with 3 to 10 amino acids of the defective gene; b) introducing each of the constructed libraries into cells to select and express individual variant proteins; And c) selecting the desired variant protein from the expressed variant protein, which exhibits faster fluorescence and darker red color than the wild type DsRed; A method of producing a red fluorescent protein FmRed (fast maturating red fluorescent protein) using an elongation mutagenesis polymerase chain reaction comprising a. A fusion protein comprising the red fluorescent protein FmRed gene according to claim 3 or 4 as a reporter or a fusion tag. A biosensor comprising a red fluorescent protein FmRed gene according to claim 3 or 4 as a reporter or a fusion tag. An operon trap vector comprising the red fluorescent protein FmRed gene according to claim 3.

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