KR101953950B1 - Development of transfer vector for targeting vacuole by signal peptide sequence - Google Patents

Abstract

The present invention relates to a recombinant vector comprising a primer of a signal transduction peptide characterized by being transported in vacuo and a nucleotide sequence encoding GFP for operably linked pYES2.

Description

 [0001] The present invention relates to a method for preparing a liquid transport vector by a signal transduction peptide,

The present invention relates to a method for confirming the activity and expression of a protein transported into a vacuole by a recombinant transport vector and a transport vector containing a signal transduction protein. More particularly, the present invention relates to a signal transduction protein isolated from a lipoprotein present in yeast , A recombinant microorganism transformed with the recombinant vector, and a method for obtaining a target protein by confirming the expression of the target protein in the lactose.

In modern science and technology, there are numerous technologies that can be commercialized, and there is interest in industries that have high added value such as pharmaceuticals and cosmetics, and are important to be distributed to people. From a molecular biological point of view, proteomics is of interest to a number of scientists, and it can be said that this is an important study that needs to be studied through both prokaryotes and eukaryotes.

All of the proteins in living organisms are constructing a signal transduction system, and research on their transport has been of interest. What signal is transmitted by the signal peptide sequence has been variously analyzed since the 1980s have. S. cerevisiae has been analyzed as a representative microorganism of eukaryotic organisms, and signal transduction peptides mainly involved in the transport of various proteins showed similarities with proteins present in cell organelles called lysosomes, which are cell organelles . This can be regarded as a factor that determines the directionality of the targeted protein, such as the transport of specific organelles or extracellular pathways. For example, when a lysosomal protein is involved in protein migration, a total of four migration methods can be cited. First, the proteins involved in the MVB pathway are numerous and many proteins have not yet been identified. The proteins transported by this pathway are generated in the golgi apparatus and transport vesicles (lysosomes) ), And then transferred to a small shear vesicle called " It is known to transport mainly membrane proteins. Next, the proteins classified into the ALP pathway are transported to the lysosome by the protein produced in the golgi apparatus. Membrane proteins are known to be transported, and proteins are transported by binding with adapter protein (AP). Third, the proteins belonging to the CVT pathway have a pathway through which proteins present in the cytosol are transported to the lysosome. Finally, the CPY pathway refers to proteins that migrate from the transport vesicle to the lysosome .

These lysosomal proteins differ in their purpose of movement by different signal sequences. The CPY pathway is the study of the pathway in which the most proteins move. A representative vesicle-like transport vesicle that is transported from the cytoplasm to the lysosome is transported to the lysosome, representing a pathway containing proteins required for transport of the carboxypeptidase Y to the lysosome. It is mainly studied at this stage because proteins of large molecules can be transported.

Selection of the transport-related signal peptide according to the protein transport pathway is performed by two representative lysosomal proteins. When the signal peptide sequence is extracted from the genomic DNA of the protein and inserted into the expression vector, Can be applied to the development of techniques for transporting desired proteins to lysosomes.

In addition, the proteins transferred to the lysosomes by the signal transduction peptide are extracted and evaluated to be utilized in the form of a vacuole in which the functions thereof are retained, and the antifungal activity and the functionality suitable for the target protein are evaluated. It can be the core of technology that the range is endless. For example, proteins that degrade aldehyde-based materials that are present only in the cytoplasm are transported to the lysosomes by signaling proteins, which results in greater efficiency in lysosomes and elimination of the use of other carriers. This is because if the lysosome extracted from the signal transduction peptide and the recombinant strain of the target protein is developed as a feed for livestock exposed to environmentally harmful diseases living in the livestock environment, the feed extracted from the environmentally friendly material can be applied to the livestock have. Therefore, we can emphasize that this technology is a back bone carrier that can transport and direct the desired protein to the desired site.

To solve this problem, the inventors isolated a signal peptide from a lipoprotein present in eukaryotic yeast cells and introduced it into a vector containing a green fluorescent protein to prepare a recombinant vector. This recombinant vector was transfected into microorganisms and the green fluorescent protein was expressed in the lysosome using the transformed microorganism. That is, according to the present invention, a carrier capable of binding to a protein existing in a cell or not existing in cells or a protein existing in the cytoplasm and transporting it to a desired cell organelle has been developed. By confirming the presence or absence of expression of a fluorescent protein, Respectively.

Accordingly, an object of the present invention is to solve the above-mentioned problems. Accordingly, an object of the present invention is to solve the above-mentioned problems, and it is an object of the present invention to provide a DNA sequence encoding a signal protein PEP4 (Proteinase A) comprising the nucleotide sequence of SEQ ID NOS: 5 and 6, Of the nucleotide sequence of the recombinant vector.

Also, an object of the present invention is to provide a recombinant vector, which comprises a base sequence encoding a signaling protein CPY (Carboxypeptide Y) consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10, and a base sequence of a target protein capable of expression in the vacuole .

It is also an object of the present invention to provide a recombinant microorganism transformed with said vector.

Also, an object of the present invention is (a) amplifying an inserted gene; (b) cleaving the amplified inserted genes with a restriction enzyme; (c) cleaving the expression vector having the target protein gene with the restriction enzyme; (d) transforming the inserted gene and an expression vector into a microorganism; And confirming the presence or absence of expression of the target protein.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Accordingly, the present invention provides a recombinant vector comprising a base sequence encoding a signal transduction protein PEP4 (Proteinase A) comprising the nucleotide sequence of SEQ ID NOS: 5 and 6, and a base sequence of a target protein capable of expression in the vacuole do.

The present invention also provides a recombinant vector comprising a base sequence encoding a signal sequence protein CPY (Carboxypeptide Y) comprising the nucleotide sequences of SEQ ID NOS: 9 and 10, and a base sequence of a target protein capable of expression in the vacuole do.

According to a preferred embodiment of the present invention, the PEP4 may be inserted into the 1st to 76th sequences of the target protein base sequence.

According to another preferred embodiment of the present invention, the CPY may be inserted into the 24th to 27th sequence of the target protein base sequence.

According to another preferred embodiment of the present invention, the signal transduction protein may be isolated from the lipoprotein present in the yeast.

According to another preferred embodiment of the present invention, the target protein may be ALD6.

The present invention also provides a recombinant microorganism transformed with said vector.

Further, the present invention provides a method for producing a transgenic plant, comprising: (a) amplifying an inserted gene; (b) cleaving the amplified inserted genes with a restriction enzyme; (c) cleaving the expression vector having the target protein gene with the restriction enzyme; (d) transforming the inserted gene and an expression vector into a microorganism; And confirming whether the target protein is expressed or not.

According to a preferred embodiment of the present invention, the inserted gene may be PEP4 or CPY.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a signal transduction peptide which is transported in vacuo and a transgenic microorganism produced by a vector into which a gene encoding a green fluorescent protein is introduced The purpose of this paper is to present the transport and orientation of target proteins through the presence or absence of green fluorescence protein expression in the organelle.

In addition, it is expected that a carrier that transports proteins through the present invention can be utilized as a drug delivery vehicle.

1 shows a DNA sequence of Proteinase A (PEP4) and a protein expression vector containing a GFP gene.
FIG. 2 shows the process of producing pYES2 :: PSP :: GFP (MBTL-DJ-1).
Fig. 3 shows a DNA sequence of Carboxypeptidase Y (CPY) and a protein expression vector containing a GFP gene.
4 shows the process of producing pYES2 :: QSP :: GFP (MBTL-DJ-2).
FIG. 5 shows a DNA expression sequence of Proteinase A (PEP4) and a protein expression vector containing ALD6 and GFP genes.
6 shows the process of producing pYES2 :: PSP :: ALD6 :: GFP (MBTL-DJ-3).
FIG. 7 shows a protein expression vector containing the DNA sequence of Carboxypeptidase Y (CPY) and the ALD6 and GFP genes.
FIG. 8 shows the process of producing pYES2 :: QSP :: ALD6 :: GFP (MBTL-DJ-4).
FIG. 9 shows fluorescence expression of MBTL-DJ-1 and MBTL-DJ-2 as recombinant yeasts as a result of fluorescence microscopy.
Fig. 10 shows fluorescence expression of recombinant yeasts, MBTL-DJ-3 and MBTL-DJ-4, as a result of fluorescence microscopy.
11 shows the results of culturing MBTL-DJ-3 and MBTL-DJ-4, extracting the vacuole and reacting with formaldehyde to obtain Vibrio This is a graph showing that the protein was completely transported through the luminescence of fischeri .

Hereinafter, the present invention will be described in more detail.

As described above, research on the transport of the target protein outside the cell organelles or cells has been continuing with increasing interest in the conventional signal transduction peptides. Accordingly, the present inventors sought to solve the above-mentioned problems by developing a carrier capable of transporting a target protein into a cell organelle and confirming the expression of a fluorescent protein.

Accordingly, the present invention provides a recombinant vector comprising a base sequence encoding a signal transduction protein PEP4 (Proteinase A) comprising the amino acid sequence of SEQ ID NOS: 5 and 6, and a base sequence of a target protein capable of expression in the vacuole do.

The present invention also provides a recombinant vector comprising a base sequence encoding a signal transduction protein CPY (Carboxypeptide Y) comprising the amino acid sequences of SEQ ID NOS: 9 and 10, and a base sequence of a target protein capable of expression in the vacuole do.

In one embodiment of the present invention, a recombinant vector was prepared as shown in the schematic diagrams shown in Figs.

The present inventors have used DNA information of each signaling protein to separate the signaling proteins PEP4 and CPY, which can be transported into the vacuole, from the eukaryotic S. cerevisiae s2805 in the process of separating the signal transduction proteins transported into specific organelles PCR was performed to confirm and confirm the signal transduction proteins in NCBI. As a result, a DNA fragment of 228 bp was obtained for the PEP4 signaling gene and a DNA fragment of 103 bp for the CPY signaling gene.

The recombinant vector may be a commercially available recombinant expression vector in which a general microbial expression vector is used as a basic framework and genes encoding PEP4 or CPY proteins are sequentially and operatively linked together with various promoters.

In the present invention, the term " expression vector " means a nucleotide sequence encoding the PEP4 protein consisting of the amino acid sequences of SEQ ID NOS: 5 and 6 according to the present invention, or a nucleotide sequence encoding the CPY protein consisting of the amino acid sequences of SEQ ID NOS: Refers to plasmids, cosmid vectors, bacteriophage vectors, viral vectors or other vectors known in the art that can be inserted or introduced, preferably the expression vector of the invention is pYES2-GFP, but is not limited thereto no.

The base sequence encoding the PEP4 protein consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6 or the CPY protein consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10 according to the present invention may be operably linked to an expression control sequence, The linked nucleotide sequence and expression control sequence may be contained within an expression vector containing a selectable marker and a replication origin. Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. &Quot; Expression control sequence " means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling transcription and translation termination.

In the present invention, the PEP4 signaling protein is inserted into the 1 to 76th amino acid sequence of the target protein base sequence. Preferably, restriction enzymes HindIII and NotI are used for the insertion, but the present invention is not limited thereto.

In the present invention, the CPY signal transduction protein is inserted into the 24th to 27th amino acid sequences of the target protein base sequence. Preferably, restriction enzymes Not I and Ear I are used for the insertion, but the present invention is not limited thereto.

In the present invention, the signal transduction protein is separated from the vacuole protein present in the yeast, and the term " vacuole " refers to a giant cell organelle surrounded by a membrane possessed by some eukaryotic cells. In addition, vacuoles can act as nutrients, wrap undesirable fragments around cells, isolate substances that may be toxic to cells, maintain fluid balance in cells called pressure, or release inappropriate substances from cells .

The present invention also provides a recombinant microorganism transformed with said vector.

The recombinant vector of the present invention can be introduced into a host cell using methods known in the art. Methods for introduction into host cells are preferably selected from the group consisting of calcium chloride method, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome mediated method liposome-mediated method) can be used.

Further, the present invention provides a method for producing a transgenic plant, comprising: (a) amplifying an inserted gene; (b) cleaving the amplified inserted genes with a restriction enzyme; (c) cleaving the expression vector having the target protein gene with the restriction enzyme; (d) transforming the inserted gene and an expression vector into a microorganism; And confirming whether the target protein is expressed or not.

First, peptides related to signal transduction in the vacuole of eukaryotic cells were isolated and amplified by amplifying the related gene region using polymerase chain reaction. Specifically, the inserted gene is PEP4 or CPY, and the gene moves according to the CPT pathway method in which proteins present in the cytoplasm are transported to the lysosome by transport vesicles.

The restriction enzyme used to make the recombinant vector of the present invention is preferably selected from two or more selected from the group consisting of HindIII, SphI I, Not I and BamHI, but is not limited thereto

The term " target protein " refers to a protein to be produced, and is not limited to a specific protein. In addition, reporter proteins may be included as the target protein, which is a protein expressed by a reporter gene, and indicates a marker protein that indicates its activity in a cell by its presence. In an embodiment of the present invention, GFP is used as the target protein. However, the target protein is not limited thereto, and any protein that can be expressed in the lump including medical and industrial proteins can be used without limitation.

In one embodiment of the present invention, it was tried to confirm whether a target protein is expressed in the vacuole of the recombinant yeast. To this end, a recombinant vector transformed with a recombinant vector in which a signal transduction protein CPY gene was inserted into the 24th to 27th amino acid sequences of the ALD6 base sequence of the target protein was prepared (FIG. 8). The vacuole of the prepared recombinant yeast was extracted and the liquid vacuole was added to a solution prepared by treating the formaldehyde with concentration to evaluate the formaldehyde reducing effect. As a result, it was confirmed that the concentration of formaldehyde was reduced by transporting the recombinant yeast ALD6 to the lysosome (Fig. 11)

Hereinafter, embodiments of the present invention will be described in detail. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  One. Saccharomyces cerevisiae on Signal transduction peptide ( signal peptide sequence ) PEP4 sequence (PSP) and GFP  Production of recombinant vectors containing together

Was used as the eukaryotic microorganism is S. cerevisiae s2805, signaling peptides (signal peptide sequence) is then DNA sequence confirmation of proteinase A (PEP4) from genebank (www.ncbi.nlm.nih.gov) of NCBI, in front of the DNA sequence PCR (Polymerase chain reaction) was carried out using SEQ ID NO: 5 and SEQ ID NO: 6 in order to amplify and secure the signal transduction peptide having 76AA. In order to obtain the GFP gene from pEGFP-C1 (BD Bioscience Clontech, USA) vector, the gene could be obtained by amplification by PCR using SEQ ID NO: 7 and SEQ ID NO: 8, and the results are shown in Table 1 below. The PCR amplification product was electrophoresed on agarose gel. The PEP4 signal peptide gene was 228 bp DNA fragment and the GFP gene was 711 bp fragment.

Primer sequence SEQ ID NO: PEP4-F1 5'-CGC AAG CTT ATG TTC AGC TTG AAA GCA T-3 ' 5 PEP4-R1 5'-TAT GCG GCC GCT TTC ACT GAA GAA AGGA -3 ' 6 GFP-F1 5'-AAT GCG GCC GCT GTG AGC AAG GGC GAG GAG -3 ' 7 GFP-R1 5'-ATT GCA TGC TTA CTT GTA CAG CTC GTC -3 ' 8

All of the above genes were purified using GeneAll PCR DNA purification kit.

Example  2. Saccharomyces cerevisiae on Signal transduction peptide ( signal peptide sequence ) QRPL (CAA ²⁴ -AGA-CCG-TTG ²⁷ ) Sequence (QSP) inserted GFP  Production of recombinant vector

The DNA information of Carboxypeptidase Y (CPY) was confirmed from NCBI's genebank (www.ncbi.nlm.nih.gov) in order to obtain a signal peptide sequence in S. cerevisiae s2805, a eukaryotic microorganism, and the GFP DNA sequence A restriction enzyme, EarI (New England Biolabs, England), was used to insert the signal transduction peptide QRPL (CAA ²⁴ -AGA-CCG-TTG ²⁷ ) into AA24-AA27. SEQ ID NO: 10 and SEQ ID NO: 11 were used to amplify the front DNA of AA ²⁴ , and SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the rear portion of AA ²⁷ was respectively amplified by PCR. . The PCR amplification product was electrophoresed on 0.7% agarose gel to obtain a 103 bp DNA fragment with a QRPL inserted as a signal peptide gene of CPY and a GFP gene with 673 bp fragment Respectively.

Primer sequence SEQ ID NO: CPY-F1 5'-TTT AGC GGC CGC ATG GTG AGC AAG GGC GAG-3 ' 9 CPY-R1 5'-ATA CTC TTC TCA ACG GTC TTT GTA CGT CGC CGT CCA GCT C -3 ' 10 CPY-F2 5'- TTT CTC TTC TTT GAA CGG CCA CAA GTT CAG C -3 ' 11 CPY-R2 5'-ATT GCA TGC TTA CTT GTA CAG CTC GTC -3 ' 12

All of the above genes were purified using GeneAll PCR DNA purification kit.

Example  3. Signal transduction peptide ( signal peptide sequence ) PEP4 sequence (PSP) and A cytoplasmic protein Aldehyde Dihydrogenase  6 ( Aldehyde dehydrogenase , ALD6 ) ≪ / RTI >

The DNA sequence of the signal peptide sequence of the proteinase A (PEP4) obtained in Example 1 was inserted in front of the cytoplasmic protein, Aldehyde dehydrogenase (ALD6), and the DNA sequence was amplified SEQ ID NO: 14 and SEQ ID NO: 15 were used in order to obtain the nucleotide sequence of SEQ ID NO. The PCR amplification products were electrophoresed on an agarose gel.

Primer sequence SEQ ID NO: PEP4-F1 5'-CGC AAG CTT ATG TTC AGC TTG AAA GCA T-3 ' 5 PEP2-R2 5'-GCT GGA TCC TTC AGT GAA GAA AGG ATG -3 ' 13 ALD6-F 5'-GGC GGA TCC ATG ACT AAG CTA CAC TTT -3 ' 14 ALD6-R 5'-GGT GCG GCC GCC AAC TTA ATT CTG ACA -3 ' 15 GFP-F2 5'-ATT GCG GCC GC GTG AGC AAG GGC GAG 3 ' 16 GFP-R2 5'-GGT GCA TGC CTT GTA CAG CTC ATC CAT? 3 ' 17

All of the above genes were purified using GeneAll PCR DNA purification kit.

Example  4. Signal transduction peptide ( signal peptide sequence ) The QRPL sequence (QSP) and A cytoplasmic protein Aldehyde Dihydrogenase  6 ( Aldehyde dehydrogenase , ALD6 ) ≪ / RTI >

(CAA ²⁴ -AGA-CCG-TTG ²⁷ ), which is a signal peptide sequence of Carboxypeptidase Y (CPY) obtained in Example 2, was designed as a primer. DNA fragments were prepared by inserting them into AA ²⁴ -AA ²⁷ of aldehyde dehydrogenase (ALD6). At this time, AA ²⁴ earlier to amplify the DNA sequence was used to SEQ ID NO: 14 and SEQ ID NO: 18, later in AA ²⁷ were obtained the products through the PCR amplification using SEQ ID NO: 19 and SEQ ID NO: 17, 0.7% Was confirmed by electrophoresis on an agarose gel. The results are shown in Table 4 below.

Primer sequence SEQ ID NO: ALD6-F 5'-GGC GGA TCC ATG ACT AAG CTA CAC TTT -3 ' 14 ACPY-R 5'-GCG CTC TTC A CAA CGG TCT TTG CTC GTA TGT CAA -3 ' 18 ACPY-F 5'-CCA CTC TTC TTT GCA ACC AAC CGG TCTA -3 ' 19 ALD6-R 5'-GGT GCG GCC GCC AAC TTA ATT CTG ACA -3 ' 15 GFP-F2 5'-ATT GCG GCC GC GTG AGC AAG GGC GAG 3 ' 16 GFP-R2 5'-GGT GCA TGC CTT GTA CAG CTC ATC CAT? 3 ' 17

All of the above genes were purified using GeneAll PCR DNA purification kit.

Example  5. Signal transduction peptides  Inserted recombinant backbone  vector( pYES2 :: PSP :: GFP , pYES2 :: QSP :: GFP, pYES2 :: PSP :: ALD6 :: GFP , pYES :: QSP :: ALD6 :: GFP )

PYES2 :: PSP :: GFP, pYES2 :: QSP: pYES2 :: PSP :: GFP in which two kinds of signal peptide sequences were inserted using yeast-protein expression vector pYES2 (Invitogen, Carlsbad, CA, USA) : GFP was prepared. Aldehyde dehydrogenase (ALD6), one of the cytoplasmic proteins, was prepared.

Example 5-1. Manufacture of MBTL-DJ-1

PYES2 :: PSP :: GFP was prepared by digesting the GFP gene DNA fragment with the PEP4 signal peptide amplified in Example 1 into HindIII and SphI and inserting the same restriction site into the cleaved pYES2 vector.

Example 5-2. Production of MBTL-DJ-2

In Example 2, pGES2 :: QSP :: GFP was prepared by inserting the CPY-derived signal peptide-inserted GFP gene DNA fragment into NotI and SphI and inserting the same restriction site into the cleaved pYES2 vector.

Example 5-3. Production of MBTL-DJ-3

The signal peptide derived from PEP4 amplified in Example 3 and the cytoplasmic protein ALD6 were synthesized together with the GFP gene. Each DNA fragment was digested with HindIII, BamHI, NotI, and SphI and inserted into a pYES2 vector To produce pYES2 :: PSP :: ALD6 :: GFP.

Example 5-4. Manufacture of MBTL-DJ-4

CPY-derived signal peptide amplified in Example 4 and ALD6, a cytoplasmic protein, were synthesized together with a GFP gene. Each DNA fragment was digested with BamHI, NotI, EarI, and SphI and inserted into a cleaved pYES2 vector pYES2 :: QSP :: ALD6 :: GFP.

Comparative Example. Production of MBTL-DJ-

a recombinant microorganism containing only the pYES2 vector was prepared.

In addition, the vectors pYES2 :: PSP :: GFP, pYES2 :: QSP :: GFP, pYES2 :: PSP :: ALD6 :: GFP, pYES :: QSP :: ALD6 :: GFP was transformed into S. cereviae s2805, a yeast strain, by electroporation, and the colonies obtained from the SD solid plate culture medium were transferred to a fresh medium to obtain a single colony. DNA sequencing method, which contains a signal peptide inserted into pYES2 :: PSP :: GFP, pYES2 :: QSP :: GFP, pYES2 :: PSP :: ALD6 :: GFP and pYES2 :: QSP :: ALD6 :: GFP The nucleotide sequence of the DNA was precisely confirmed.

Example  6. To signal transduction peptides  due to into the vacuole  Expression of the transfected protein

The recombinant microorganism prepared in Example 5 was inoculated in 10 mL of SD liquid medium (6.7 g / L of yeast nitrogen base, 5 g / L of amino acid and 20 g / L of glucose) Lt; / RTI > until shaking at -0.8. After removing the microorganism from the resulting culture, add 15 mL of SG liquid medium (yeast nitrogen base 6.7 g / L, amino acid 5 g / L and galactose 20 g / L). 6.67 mL of the thus-obtained culture (OD value: 0.4) was added to 50 mL of SG liquid medium and incubated at 30 DEG C and 160 rpm for 10 hours. LysoTracker, which specifically reacts with cultured cells, was treated with LysoTracker to examine the expression level of the target protein and whether the protein was well targeted in the lysosome. Fluorescence microscopy confirmed that the protein was overexpressed, and Merged The lysosomes in the lysosomes were activated. As a result of MBTL-DJ-1 and MBTL-DJ-2, it can be confirmed that the GFP protein, which is not a cell-possessing protein, is transported into the yeast vacuole by a signal transduction peptide. MBTL-DJ-3 and MBTL As a result of -DJ-4, it was confirmed that the ALD6 protein expressed in the cytoplasm was transported into the yeast vacuole by the signal transduction peptide.

Example  7. To signal transduction peptides  due to into the vacuole  Transported Aldehyde Dihydrogenase  6 ( Aldehyde dehydrogenase , ALD6 )

The vacuole was extracted from the recombinant yeast (MBTL-DJ-3, MBTL-DJ-4) prepared in Example 5, and the formaldehyde reducing effect by the extracted vacuole was evaluated.

The recombinant yeast (MBTL-DJ-3, MBTL-DJ-4) prepared in Example 5 was shake cultured under the proposed culture conditions. The cells were then pulverized with an ultrasonic disrupter in galactose-derived recombinant yeast and the vacoule was recovered by centrifugation.

The bioluminescence, vibrio fischeri were cultivated in LB medium for 12 hours to 15 hours, cultured for 2 hours to 3 hours in the present culture, cultured to have an OD (Optical Density) value of 0.4 to 0.6, Is added to the solution treated with concentration (62.5 ppm, 125 ppm, 250 ppm, 500 ppm) and reacted for 1 hour to 1 hour and 30 minutes, thereby confirming reduction of formaldehyde.

This reduces the formaldehyde that ALD6 is transported to the lysosome of the recombinant yeast MBTL-DJ-3 and MBTL-DJ-4 of the present invention vibrio fischeri , which can be determined by comparing the degree of light emitted by a luminometer.

As shown in Fig. 11, MBTL-DJ-3 and MBTL-DJ-4 exhibited significantly higher levels of formaldehyde than those of the control group at concentrations of 63 ppm to 500 ppm. That is, the control group with increasing the concentration of formaldehyde is a decrease in the survival rate of vibrio fischeri decreased the light emission amount of Luminescene, in the case of the recombinant yeast MBTL-DJ-3 and MBTL-DJ-4 include formaldehyde concentration 63ppm To 125 ppm, Luminescene decreased or rather increased. Therefore, it can be confirmed that the target protein ALD6 is migrated into the yeast cell membrane by the signal peptide.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> A recombinant vector for expressing, signal protein transformed          by the recombinant vector and Biosensor detecting signal peptide          sequence <130> 1060573 <160> 23 <170> Kopatentin 2.0 <210> 1 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> signal peptide sequence for PEP4 (PSP) <400> 1 atgttcagct tgaaagcatt attgccattg gccttgttgt tggtcagcgc caaccaagtt 60 gctgcaaaag tccacaaggc taaaatttat aaacacgagt tgtccgatga gatgaaagaa 120 gtcactttcg agcaacattt agctcattta ggccaaaagt acttgactca atttgagaaa 180 gctaaccccg aagttgtttt ttctagggag catcctttct tcactgaa 228 <210> 2 <211> 12 <212> DNA <213> Artificial Sequence <220> Signal peptide sequence for CPY (QSP) <400> 2 caacggtctt tg 12 <210> 3 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> sequence for GFP <400> 3 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720                                                                          720 <210> 4 <211> 1503 <212> DNA <213> Artificial Sequence <220> <223> signal peptide sequence for ALD6 <400> 4 atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taa 1503 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PEP4-F1 (forward primer) <400> 5 cgcaagctta tgttcagctt gaaagcat 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > PEP4-R1 (reverse primer) <400> 6 tatgcggccg ctttcactga agaaagga 28 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> GFP-F1 (forward primer) <400> 7 aatgcggccg ctgtgagcaa gggcgaggag 30 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> GFP-R1 (reverse primer) <400> 8 attgcatgct tacttgtaca gctcgtc 27 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CPY-F1 (forward primer) <400> 9 tttagcggcc gcatggtgag caagggcgag 30 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> CPY-R1 (reverse primer) <400> 10 atactcttct caacggtctt tgtacgtcgc cgtccagctc 40 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> CPY-F2 (forward primer) <400> 11 tttctcttct ttgaacggcc acaagttcag c 31 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> CPY-R2 (reverse primer) <400> 12 attgcatgct tacttgtaca gctcgtc 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > PEP4-R2 (reverse primer) <400> 13 gctggatcct tcagtgaaga aaggatg 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> ALD6-F (forward primer) <400> 14 ggcggatcca tgactaagct acacttt 27 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> ALD6-R (reverse primer) <400> 15 ggtgcggccg ccaacttaat tctgaca 27 <210> 16 <211> 2268 <212> DNA <213> Artificial Sequence <220> <223> GFP-F2 (forward primer) <400> 16 ggcggatcca tgactaagct acactttgac actgctgaac cagtcaagat cacacttcca 60 aatggtttga catacgagca aagaccgcca ctcttctttg caaccaaccg gtctattcat 120 taacaacaag tttatgaaag ctcaagacgg taagacctat cccgtcgaag atccttccac 180 tgaaaacacc gtttgtgagg tctcttctgc caccactgaa gatgttgaat atgctatcga 240 atgtgccgac cgtgctttcc acgacactga atgggctacc caagacccaa gagaaagagg 300 ccgtctacta agtaagttgg ctgacgaatt ggaaagccaa attgacttgg tttcttccat 360 tgaagctttg gacaatggta aaactttggc cttagcccgt ggggatgtta ccattgcaat 420 caactgtcta agagatgctg ctgcctatgc cgacaaagtc aacggtagaa caatcaacac 480 cggtgacggc tacatgaact tcaccacctt agagccaatc ggtgtctgtg gtcaaattat 540 tccatggaac tttccaataa tgatgttggc ttggaagatc gccccagcat tggccatggg 600 taacgtctgt atcttgaaac ccgctgctgt cacaccttta aatgccctat actttgcttc 660 tttatgtaag aaggttggta ttccagctgg tgtcgtcaac atcgttccag gtcctggtag 720 aactgttggt gctgctttga ccaacgaccc aagaatcaga aagctggctt ttaccggttc 780 tacagaagtc ggtaagagtg ttgctgtcga ctcttctgaa tctaacttga agaaaatcac 840 tttggaacta ggtggtaagt ccgcccattt ggtctttgac gatgctaaca ttaagaagac 900 tttaccaaat ctagtaaacg gtattttcaa gaacgctggt caaatttgtt cctctggttc 960 tagaatttac gttcaagaag gtatttacga cgaactattg gctgctttca aggcttactt 1020 ggaaaccgaa atcaaagttg gtaatccatt tgacaaggct aacttccaag gtgctatcac 1080 taaccgtcaa caattcgaca caattatgaa ctacatcgat atcggtaaga aagaaggcgc 1140 caagatctta actggtggcg aaaaagttgg tgacaagggt tacttcatca gaccaaccgt 1200 tttctacgat gttaatgaag acatgagaat tgttaaggaa gaaatttttg gaccagttgt 1260 cactgtcgca aagttcaaga ctttagaaga aggtgtcgaa atggctaaca gctctgaatt 1320 cggtctaggt tctggtatcg aaacagaatc tttgagcaca ggtttgaagg tggccaagat 1380 gttgaaggcc ggtaccgtct ggatcaacac atacaacgat tttgactcca gagttccatt 1440 cggtggtgtt aagcaatctg gttacggtag agaaatgggt gaagaagtct accatgcata 1500 cactgaagta aaagctgtca gaattaagtt ggcggccgca ccggccgcag tgagcaaggg 1560 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 1620 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct 1680 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct 1740 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt 1800 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg 1860 cactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 1920 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 1980 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa 2040 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca 2100 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 2160 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 2220 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggcatg 2268 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> GFP-R2 (reverse primer) <400> 17 ggtgcatgcc ttgtacagct catccat 27 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> ACPY-R (reverse primer) <400> 18 gcgctcttca caacggtctt tgctcgtatg tcaa 34 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > ACPY-F (forward primer) <400> 19 ccactcttct ttgcaaccaa ccggtcta 28 <210> 20 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> sequence for PSP :: GFP <400> 20 atgttcagct tgaaagcatt attgccattg gccttgttgt tggtcagcgc caaccaagtt 60 gctgcaaaag tccacaaggc taaaatttat aaacacgagt tgtccgatga gatgaaagaa 120 gtcactttcg agcaacattt agctcattta ggccaaaagt acttgactca atttgagaaa 180 gctaaccccg aagttgtttt ttctagggag catcctttct tcactgaagt gagcaagggc 240 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 300 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 360 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 420 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 480 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 540 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 600 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 660 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 720 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 780 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 840 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 900 accgccgccg ggatcactct cggcatggac gagctgtaca agtaa 945 <210> 21 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> Sequence for QSP :: GFP <400> 21 ggccgcatgg tgagcaaggg cgagctgttc accggggtgg tgcccatcct ggtcgagctg 60 gacggcgacg tacaaagacc gttgaacggc cacaagttca gcgtgtccgg cgagggcgag 120 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 180 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 240 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 300 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 360 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 420 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 480 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 540 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 600 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 660 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 720 gagctgtaca agtaa 735 <210> 22 <211> 2227 <212> DNA <213> Artificial Sequence <220> <223> Sequence for PSP :: ALD6 :: GFP <400> 22 gatccatgac taagctacac tttgacactg ctgaaccagt caagatcaca cttccaaatg 60 gtttgacata cgagcaacca accggtctat tcattaacaa caagtttatg aaagctcaag 120 acggtaagac ctatcccgtc gaagatcctt ccactgaaaa caccgtttgt gaggtctctt 180 ctgccaccac tgaagatgtt gaatatgcta tcgaatgtgc cgaccgtgct ttccacgaca 240 ctgaatgggc tacccaagac ccaagagaaa gaggccgtct actaagtaag ttggctgacg 300 aattggaaag ccaaattgac ttggtttctt ccattgaagc tttggacaat ggtaaaactt 360 tggccttagc ccgtggggat gttaccattg caatcaactg tctaagagat gctgctgcct 420 atgccgacaa agtcaacggt agaacaatca acaccggtga cggctacatg aacttcacca 480 ccttagagcc aatcggtgtc tgtggtcaaa ttattccatg gaactttcca ataatgatgt 540 tggcttggaa gatcgcccca gcattggcca tgggtaacgt ctgtatcttg aaacccgctg 600 ctgtcacacc tttaaatgcc ctatactttg cttctttatg taagaaggtt ggtattccag 660 ctggtgtcgt caacatcgtt ccaggtcctg gtagaactgt tggtgctgct ttgaccaacg 720 acccaagaat cagaaagctg gcttttaccg gttctacaga agtcggtaag agtgttgctg 780 tcgactcttc tgaatctaac ttgaagaaaa tcactttgga actaggtggt aagtccgccc 840 atttggtctt tgacgatgct aacattaaga agactttacc aaatctagta aacggtattt 900 tcaagaacgc tggtcaaatt tgttcctctg gttctagaat ttacgttcaa gaaggtattt 960 acgacgaact attggctgct ttcaaggctt acttggaaac cgaaatcaaa gttggtaatc 1020 catttgacaa ggctaacttc caaggtgcta tcactaaccg tcaacaattc gacacaatta 1080 tgaactacat cgatatcggt aagaaagaag gcgccaagat cttaactggt ggcgaaaaag 1140 ttggtgacaa gggttacttc atcagaccaa ccgttttcta cgatgttaat gaagacatga 1200 gaattgttaa ggaagaaatt tttggaccag ttgtcactgt cgcaaagttc aagactttag 1260 aagaaggtgt cgaaatggct aacagctctg aattcggtct aggttctggt atcgaaacag 1320 aatctttgag cacaggtttg aaggtggcca agatgttgaa ggccggtacc gtctggatca 1380 acacatacaa cgattttgac tccagagttc cattcggtgg tgttaagcaa tctggttacg 1440 gtagagaaat gggtgaagaa gtctaccatg catacactga agtaaaagct gtcagaatta 1500 agttgtaagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 1560 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 1620 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 1680 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 1740 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 1800 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 1860 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 1920 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 1980 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 2040 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 2100 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 2160 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 2220 aggcatg 2227 <210> 23 <211> 2268 <212> DNA <213> Artificial Sequence <220> <223> Sequence for QSP :: ALD6 :: GFP <400> 23 ggcggatcca tgactaagct acactttgac actgctgaac cagtcaagat cacacttcca 60 aatggtttga catacgagca aagaccgcca ctcttctttg caaccaaccg gtctattcat 120 taacaacaag tttatgaaag ctcaagacgg taagacctat cccgtcgaag atccttccac 180 tgaaaacacc gtttgtgagg tctcttctgc caccactgaa gatgttgaat atgctatcga 240 atgtgccgac cgtgctttcc acgacactga atgggctacc caagacccaa gagaaagagg 300 ccgtctacta agtaagttgg ctgacgaatt ggaaagccaa attgacttgg tttcttccat 360 tgaagctttg gacaatggta aaactttggc cttagcccgt ggggatgtta ccattgcaat 420 caactgtcta agagatgctg ctgcctatgc cgacaaagtc aacggtagaa caatcaacac 480 cggtgacggc tacatgaact tcaccacctt agagccaatc ggtgtctgtg gtcaaattat 540 tccatggaac tttccaataa tgatgttggc ttggaagatc gccccagcat tggccatggg 600 taacgtctgt atcttgaaac ccgctgctgt cacaccttta aatgccctat actttgcttc 660 tttatgtaag aaggttggta ttccagctgg tgtcgtcaac atcgttccag gtcctggtag 720 aactgttggt gctgctttga ccaacgaccc aagaatcaga aagctggctt ttaccggttc 780 tacagaagtc ggtaagagtg ttgctgtcga ctcttctgaa tctaacttga agaaaatcac 840 tttggaacta ggtggtaagt ccgcccattt ggtctttgac gatgctaaca ttaagaagac 900 tttaccaaat ctagtaaacg gtattttcaa gaacgctggt caaatttgtt cctctggttc 960 tagaatttac gttcaagaag gtatttacga cgaactattg gctgctttca aggcttactt 1020 ggaaaccgaa atcaaagttg gtaatccatt tgacaaggct aacttccaag gtgctatcac 1080 taaccgtcaa caattcgaca caattatgaa ctacatcgat atcggtaaga aagaaggcgc 1140 caagatctta actggtggcg aaaaagttgg tgacaagggt tacttcatca gaccaaccgt 1200 tttctacgat gttaatgaag acatgagaat tgttaaggaa gaaatttttg gaccagttgt 1260 cactgtcgca aagttcaaga ctttagaaga aggtgtcgaa atggctaaca gctctgaatt 1320 cggtctaggt tctggtatcg aaacagaatc tttgagcaca ggtttgaagg tggccaagat 1380 gttgaaggcc ggtaccgtct ggatcaacac atacaacgat tttgactcca gagttccatt 1440 cggtggtgtt aagcaatctg gttacggtag agaaatgggt gaagaagtct accatgcata 1500 cactgaagta aaagctgtca gaattaagtt ggcggccgca ccggccgcag tgagcaaggg 1560 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 1620 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct 1680 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct 1740 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt 1800 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg 1860 cactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 1920 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 1980 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa 2040 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca 2100 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 2160 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 2220 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggcatg 2268

Claims (9)

As a recombinant expression vector represented by a cleavage map in Fig. 7,
A nucleotide sequence of SEQ ID NO: 2 encoding a signal peptide sequence of Carboxypeptidase Y (CPY); And
A nucleotide sequence of SEQ ID NO: 4 which codes for aldehyde dehydrogenase (ALD6); / RTI &gt;
Wherein the signal peptide sequence of Carboxypeptidase Y (CPY) is inserted between the 23rd amino acid residue and the 24th amino acid residue of the aldehyde dehydrogenase 6.
2. The recombinant vector according to claim 1, wherein the base sequence of QSP :: ALD6 :: GFP of the recombinant expression vector is represented by the nucleotide sequence of SEQ ID NO: 23.
A transformant transformed with the expression vector of claim 1.
(a) a first primer set forth in SEQ ID NO: 14 and a primer set of a reverse primer set forth in SEQ ID NO: 18 are used to amplify the first to 23rd amino acid residues and the CPY signaling protein of ADL6, Amplifying a fragment of a base sequence encoding an amino acid residue;
(b) a nucleotide sequence encoding the 27th amino acid residue of the signal transduction protein of CPY using the forward primer represented by SEQ ID NO: 19 and the primer set of the reverse primer represented by SEQ ID NO: 17; A fragment of the nucleotide sequence encoding the 24th amino acid residue to the 27th amino acid residue of ADL6; And amplifying a fragment of the base sequence encoding the amino acid of GFP;
(c) cleaving the amplified genes with a restriction enzyme;
(d) trimming the expression vector with the restriction enzyme; And
(e) cloning the gene and an expression vector to transform the microorganism.
delete delete delete delete delete

Images