WO2016122058A1 - Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds - Google Patents

Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds Download PDF

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WO2016122058A1
WO2016122058A1 PCT/KR2015/006854 KR2015006854W WO2016122058A1 WO 2016122058 A1 WO2016122058 A1 WO 2016122058A1 KR 2015006854 W KR2015006854 W KR 2015006854W WO 2016122058 A1 WO2016122058 A1 WO 2016122058A1
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pah
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박영식
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인제대학교 산학협력단
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    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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    • C12Y104/0102—Phenylalanine dehydrogenase (1.4.1.20)

Definitions

  • the present invention relates to a method for analyzing the activity of human Phenylalanine Hydroxylase (PAH) using a cellular bacterium, specifically transforming a PAH gene containing a phenylketonuria-related mutation to a cellular bacterium, a minimal nutrient medium tyrosine deficient After culturing at, the present invention relates to a method for analyzing human mutant PAH activity by measuring the growth rate of cellular slime molds.
  • PAH Phenylalanine Hydroxylase
  • Phenylalanine hydroxylase (PAH; EC 1.14.16.1) is an enzyme containing iron ions (Fe 2+ ), coenzyme tetrahydrobioterin (6R-L-erythro-tetrahydrobiopterin (BH4)) and molecular oxygen (O 2). ) Catalyzes the conversion of L-phenylalanine to L-tyrosine in the presence of In humans, PAH is present in many liver and brain tissues, and is mainly involved in the metabolism of phenylalanine, which is consumed as a food in the liver, and is important for the synthesis of neurotransmitters such as dopamine in the brain (Fitzpatrick PF. 2012 Arch Biochem Biophys 519, 194201) .
  • PKU phenylketonuria
  • HPA hyperphenylalaninemia
  • PKU is divided into mild PKU (600-900 ⁇ M), moderate PKU (900-1200 ⁇ M), and classic PKU (> 1200 ⁇ M), all requiring compulsory treatment (Heintz C, et al. 2013 Hum Mutat 34, 927-936).
  • BH4 The pharmacological mechanism of BH4 is known because BH4 inhibits the activation of enzymes by L-Phe (called 'pharmacological chaperone').
  • L-Phe L-Phe
  • BH4 does not work effectively in all PKU patients, studies are underway to find pharmacological chaperones that increase PAH stability like BH4, and patient-specific therapies have been proposed (Underhaug J, et al. 2012 Curr Top Med). Chem 12, 2534-2545).
  • an object of the present invention is to provide a method for analyzing human mutant PAH activity by measuring the growth rate of a cellular bacterium transformed with a PAH gene including a phenylketonuria-related mutation as a cell-level analysis system.
  • Another object of the present invention is to provide a method for screening a phenylketonuria drug using the transformed cellular slime mold.
  • PAH human phenylalanine hydroxylase
  • the PAH activity analysis system using various expression systems has a high reliability and reliability in the analysis process due to protein stability, serious differences between in vivo and in vitro phenotype.
  • E. coli E. coli, yeast cells, mammalian cell lines and human cells
  • an expression system that can analyze the phenotype of human PAH gene mutations at the cellular level using cellular bacteria.
  • human PAH cDNA containing each of the various mutations known to cause phenylketonuria, including wild type was inserted into an expression vector, expressed in a transformant of the PAH gene-substituted cellular slime mold, and grown at the minimum medium (FM medium).
  • FM medium minimum medium
  • the cellular slime mold produces very high levels of L-erythro-tetrahydrobiopterin (BH4) and its isomer D-threo-BH4 (DH4) compared to human cells (the two are called tetrahydroterin). They act as a coenzyme of PAH and as a so-called pharmacological chaperone that contributes to protein stability. Therefore, cellular slime molds exert their pharmacological chaperone effect without adding them extracellularly.
  • BH4 L-erythro-tetrahydrobiopterin
  • DH4 isomer D-threo-BH4
  • tetrahydro sepia aminopterin reductase (sepiapterin reductase) gene is mutated cells Slime Molds substituted (spr -) involved in the biosynthesis of aminopterin was expressed in the mutant PAH.
  • Slime Molds substituted (spr -) involved in the biosynthesis of aminopterin was expressed in the mutant PAH.
  • the decrease in the amount of PAH protein was confirmed in the case of F39L, K42I, and I65T, which are known to be mutations in response to the BH4 drug among patients with phenylketonuria having a defect in the PAH gene.
  • This demonstrates that the cellular slime mold can verify the pharmacological chaperone effect of BH4 and that it can be used to develop other pharmacological chaperones.
  • the 'cellular slime mold' is a fungi belonging to a river of eukaryotic subtype cell slime molds, mononuclear amoeba-like cells are collected to form a junctional variant and later form an accumulation body. Each of the collected cells is completely independent of the whole life history, and is thought to be closer to the protozoa, amoeba, rather than fungi. Dictyiostelium discoidem , one of the cellular bacteria , is a species whose life history is well known.
  • the cell slime mold is known to be useful for eukaryotic protein expression as a research model organism that is important for biomedical research (Arya R, et al.
  • the PAH of the cellular bacilli has a structure and amino acid sequence very similar to that of human PAH. As with human PAH, each homomer as an isomeric tetramer enzyme is divided into three domains: an amino terminal regulatory domain; a catalytic domain to which a substrate and a coenzyme bind; and a carboxy terminal multimeric domain. Interestingly, the cellular bacillus produces a large amount of the isomer D-threo-isomer (DH4) in addition to BH4 (Kim HL, et al.
  • DH4 isomer D-threo-isomer
  • the gene encoding the human PAH may have any base sequence encoding a human phenylalanine hydroxylase (hPAH) protein, but preferably a base encoding the amino acid sequence of SEQ ID NO: 1 Sequence, and more preferably hPAH cDNA (ORF) having a nucleotide sequence of SEQ ID NO: 2.
  • hPAH human phenylalanine hydroxylase
  • ORF hPAH cDNA
  • the “mutation causing phenylketonuria” is due to a lack of enzymatic activity or gene expression inhibition due to nucleotide changes such as substitution, deletion and insertion of bases in a gene encoding PAH protein. All mutations that cause phenylketonuria are included. Since the present invention has a technical feature in that the cellular slime mold is used to analyze the activity of such mutant PAH, the mutant PAH used for transforming the cellular slime mold is not particularly limited.
  • the PAH gene mutations identified so far are 852 (BioPKU.org), two-thirds of which are missense mutations with amino acid changes. (http://www.pahdb.mcgill.ca/).
  • the mutation is a missense selected from the group consisting of amino acid positions F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W in the amino acid sequence of human PAH represented by SEQ ID NO: 1 It's a mutation.
  • the term 'missense mutation' refers to a type of mutation that causes one base of the DNA to be replaced with another base to change the codon of an amino acid to another codon. -directed mutagenesis).
  • the gene sequence encoding human PAH protein is used as a starting point for the generation of mutations selected in the present invention.
  • One skilled in the art can optionally use various known standard site-directed mutagenesis methods for mutagenesis of the amino acid positions listed above (Sambrook, J. et al. (1989), supra).
  • One commonly used method is to introduce mutations using PCR using a synthetic oligonucleotide mixture.
  • the missense mutation can be carried out by replacing the base of the codon using a primer represented by SEQ ID NO: 3 to 18.
  • PAH gene in which such a mutation is induced can be inserted into an appropriate expression vector and transformed into a host cell, a cellular bacterium.
  • the term 'vector' refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked, and one type of vector, 'plasmid', is a circular double-stranded DNA that can additionally connect DNA fragments therein. It means a loop.
  • the term 'expression vector' refers to a vector that directs the expression of a gene encoding a target protein that is operably linked as a recombinant vector capable of expressing a target protein in a suitable host cell.
  • the expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector such as an adenovirus vector, a retroviral vector, and the like, and preferably in the use of recombinant DNA technology. .
  • the term 'transformation' refers to a phenomenon in which DNA is introduced into a host so that DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration, thereby introducing an external DNA into a cell and causing an artificial genetic change. it means.
  • the host cell of the present invention is a cellular bacteria, and in one embodiment of the present invention, Dictyostelium discoideum was used as a representative example of the cellular bacteria.
  • any transformation method may be used, and may be easily performed according to conventional methods in the art.
  • the Hanahan method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, and silicon carbide which have improved efficiency by using a CaCl 2 precipitation method and a reducing material called DMSO (dimethyl sulfoxide) in the CaCl 2 method Agitation with fibers, agrobacterial mediated transformation, transformation with PEG, dextran sulfate, lipofectamine mediated transformation, and the like. Therefore, the transformant can be obtained by introducing the expression vector including the mutated PAH gene of the present invention into a cellular bacterium using the transformation method without limitation in the present invention.
  • the growth rate of the cellular mycobacteria is characterized by a linear correlation with PAH activity.
  • a transformant containing a mutant cytoplasmic bacterium ( pah ⁇ ) substituted with a PAH gene, a wild-type cellular bacterial (WT), and an expression vector into which a mutant human PAH gene of the present invention is inserted respectively
  • the growth rate and the level of PAH activity for each transformant showed a correlation.
  • a linear regression analysis to confirm that there is a close correlation between the growth rate of the cellular slime mold and PAH activity, as shown in B of FIG. 1, a statistically significant linear correlation was confirmed.
  • the candidate substance is determined as pharmacological saffron for PAH, phenylketonuria using the cellular bacterium ( phenylketonuria) provides a method for screening a therapeutic agent.
  • the recombinant vector is a recombinant vector having a mutated hPAH gene inserted into a pDXA-3H vector and has a cleavage map as shown in FIG. 5.
  • the “pharmacological chaperone” has been found to be a protein misfolding disease in which phenylketonuria (PKU) is mainly caused by abnormalities in protein folding (Heintz C, et al. 2013 Hum Mutat 34, 927 936), especially in the case of missense mutant PAHs, which contribute to protein folding or correct misfolded proteins because of the problem of protein folding, which reduces protein stability and thereby promotes protein degradation. And substances or small molecules that help to restore function.
  • Korean Patent Publication No. 10-2007-0005550 discloses a method of administering a substance such as BH4 for the treatment of PKU.
  • BH4 does not work effectively in all PKU patients, studies are underway to find pharmacological chaperones that increase PAH stability like BH4.
  • the pharmacological chaperone candidate in step 2) may be a natural compound, a synthetic compound, an enzyme, a protein or a nucleic acid.
  • the candidate substance By measuring the growth rate of the cellular mycobacteria cultured in the medium to which the candidate is added, the candidate substance can be judged as a pharmacological saffron for PAH by observing how the candidate substance affects the activity of the mutant PAH. .
  • the candidate can be expected to play a similar role as the pharmaceutical chaperone for mutant PAH. In such cases the candidates can be expected to improve the protein stability of the mutant PAH.
  • the present invention has the following effects and advantages.
  • Cell-level mass spectrometry systems quantitative analysis using growth rates, especially for structurally unstable mutant PAH.
  • Protein level analysis system mutant PAHs can be studied without purification.
  • A growth rate and PAH activity. Growth rate is expressed as a percentage of wild type (WT).
  • B Quantitative correlation between PAH activity and growth rate. Sigma plots were used for data analysis. The correlation coefficient (r2) is given as 95% confidence intervals (dotted lines).
  • Figure 2 is a result showing the effect on intracellular tetrahydropteridine levels and nutrient medium on hPAH.
  • A Results of Western blot analyzed by chemiluminescent reaction: I, pah - cells cultured in FM medium; II, spr - cells cultured in FM medium; III, pah - cells cultured in HL5 medium. 50 ⁇ g of total protein and the same crude extract were analyzed by 12.5% SDS-PAGE and Western blotting.
  • B The amount of PAH residual protein in pah - and spr - cells.
  • C Amount of residual PAH protein in pah - cells cultured in FM and HL5 medium.
  • D PAH activity measured from pah - cells cultured in FM and HL5 medium. All data are expressed as percentage of wild type (WT).
  • FIG. 3 shows the relationship between PAH activity and protein levels of hPAHs expressed in the pah ⁇ strain.
  • the mean data in FIG. 1B and FIG. 2 are expressed as percentage of wild type.
  • Figure 5 shows a cleavage map of the recombinant vector into which the mutant hPAH gene was introduced according to the present invention.
  • Wild-type Cytomegalovirus ( Dictyostelium) discoideum Ax2 Dictybase (purchased from http://dictybase.org/ ) stock center is HL5 medium (10 g glucose per liter, 5 g yeast extract, 10 g protease peptone, 0.35 g KH 2 PO 4 , 0.35 g Na 2 HPO Incubated with 100 ⁇ g / ml of streptomycin sulfate and 100 U / ml of penzylpenicillin potassium to 4 12 H 2 O, pH6.4 (Watts DJ et al. 1970 Biochem J 119, 171174). ). Previous study (Kim HL, et al. 2012, FEBS Lett .
  • the PAH knockout mutant (pah which has through-) and sepia aminopterin reductase knockout mutant (spr -) was cultured by the addition of Blasticidine S of 10 ⁇ g / ml in HL5 medium.
  • Mutants transformed with human PAH cDNA were maintained in medium supplemented with 10 ⁇ g / ml Blasticidine S and G418 respectively.
  • the pah - and pr - has a resistance gene for G418-resistant gene is inserted into the chromosome via recombination, hPAH-pDXA-3H expression vectors for Blasticidine S is inserted.
  • Mutants were also cultured in FM minimal culture medium (ForMedium TM , UK).
  • the overlap extension PCR method was used to replace amino acid residues located at 39, 42, 48, 65, 252, 255, 349, 408 on the amino acid sequence of human PAH represented by SEQ ID NO: 1 (Heckman KL et al., 2007 Nat Protocols 2, 924-932).
  • Two complementary PCR primers, each containing a base sequence to be substituted, were prepared as shown in Table 1 below.
  • PCR reactions were performed using 1 ⁇ reaction buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 mM MgCl 2 , 0.2 mMd NTPs, 0.5 pmole primer, appropriate amount of template DNA, 2 units.
  • pfu DNA polymerase was added to final 50 ⁇ l.
  • the DNA was denatured for the first time at 95 ° C. for 5 minutes, and then amplified repeatedly for 1 minute at 95 ° C., 1 minute at 62 ° C. and 1 minute at 72 ° C. Finally, the PCR reaction was terminated by giving an extended time of 10 minutes at 72 °C.
  • the amplified DNA was confirmed by electrophoresis on a 0.7% agarose gel.
  • Mutant hPAH gene was isolated by treatment with KpnI / NsiI restriction enzyme and inserted into pDXA-3H vector (Manstein DJ, et al. 1995 Gene 162, 129134). Cells were recovered by centrifuging the cell cultures previously cultured at 350 ⁇ g for 3 minutes at 4 ° C. The recovered cells were washed twice with cold electroporation buffer (20 mM HEPES, 50 mM KCl, 10 mM NaCl, 1 mM MgSO 4 , 5 mM NaHCO 3 , 1 mM NaH 2 PO 4 , pH7.0). After removing the medium, the cells were suspended in an electroporation buffer at a concentration of 5 ⁇ 10 6 cells.
  • the transformed cells were first incubated in HL5 medium to 2 ⁇ 10 6 cells / ml, washed with FM medium, and then inoculated in FM medium at a concentration of 1 ⁇ 10 6 cells / ml. After culturing for 2 days in a shaking incubator at 22 ° C. and 150 rpm, the cell number was measured using a hemacytometer, and the cells were recovered by centrifugation at 8,000 rpm for 5 minutes.
  • the recovered cells were suspended in 100 ⁇ l of lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM PMSF). After freezing and thawing three times using liquid nitrogen to crush the cells and centrifuged for 20 minutes at 4 °C, 15,000 rpm to recover the supernatant (coenzyme extract).
  • the recovered coenzyme extract was recovered protein using a Sephadex G-25 spin column. The concentration of protein was measured by Bradford method and BSA (bovine serum albumin) was used as a standard protein.
  • PAH activity assay was performed by adding 100 mM Tris-HCl (pH 7.5), 2 mM L-phenylalanine, 100 unit catalase, 5 mM DTT, 0.4 mM BH4, 10 ⁇ g of coenzyme extract to 50 ⁇ l of the reaction solution for 10 minutes at 37 ° C. After the reaction, the reaction was stopped with the same amount of 5% (v / v) Trichloroacetic Acid solution (Kim HL, et al, 2012 FEBS Lett 586, 3596-3600). The amount of L-tyrosine present in the supernatant by centrifugation at 13,000 rpm for 10 minutes was quantified using HPLC.
  • Coenzyme extract 50 ⁇ g was mixed with 5X sample buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 30% glycerol, 5% ⁇ -mercapitalethanol, 0.02% bromophenol blue), followed by SDS-PAGE and Western blotting. It was. After SDS-PAGE, proteins were transferred to nitrocellulose membranes by electrophoresis at 45 volt for 2 hours using transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 10% Methanol).
  • 5X sample buffer 250 mM Tris-HCl pH 6.8, 10% SDS, 30% glycerol, 5% ⁇ -mercapitalethanol, 0.02% bromophenol blue
  • the transferred membrane was washed with 10 ml of TTBS (1X TBS-10 mM Tris, 150 mM NaCl, pH 7.5 added 500 ⁇ l Tween-20) solution, and 10 ml of blocking (5 in 10 ml TTBS solution). The reaction was stirred gently for 1 hour with a solution). After washing with 10 ml of TTBS solution, 5 ⁇ l of primary antibody (human PAH antibody; Abcam) was added to 10 ml of TTBS solution, left overnight with gentle shaking, and then washed twice with 10 ml of TTBS solution for 5 minutes. .
  • PAH gene is substituted with mutant cells Slime Molds (pah -) as well as wild-type to the created transformants 9 kinds of each mutant human PAH gene inserted into the expression vector. These transformants were grown in FM medium for 48 hours and then growth rate was compared (A in FIG. 1).
  • the pah ⁇ strain was not able to grow at all in the FM medium, but the wild type strain (WT) as well as the mutant transformants were all grown to some extent.
  • WT wild type strain
  • S349L transformants known as mutations that cause severe PKU, showed the lowest growth, and PAH activity was barely measured.
  • growth rate and level of PAH activity were correlated with each transformant, suggesting that the level was related to the severity of PKU symptoms. This shows a close correlation between growth rate and PAH activity. A linear regression analysis was performed to confirm this.
  • the protein amount was varied according to the mutation as expected.
  • K42I, L48S, S349L, and R408W were less than 10% of wild type, and other mutations were more than 50%.
  • the difference in protein amount by mutation means that human PAH is not subject to random degradation as an exogenous protein in cellular bacteria, but faces a similar problem in protein folding as in human cells.
  • mutant PAH proteins are presumed to have the pharmacological chaperone effect by them. To verify this effect, mutant PAH proteins were expressed in mutant strains ( spr ⁇ ) deficient in sepiaterin reductase. Since the PAH of the cellular mycobacteria remained in this strain, only the protein amount of human PAH was analyzed.
  • the S349L strain was grown in the FM medium to which the yeast extract was added, and then Western blot analysis was performed.
  • 4 is a Western blot result, the photo at the top shows the result of the Western blot and the graph at the bottom shows the quantitative value.
  • the concentration of yeast extract 1 ⁇ was 5 g per liter. According to the results shown in Figure 4, it was confirmed that there is a component in the yeast extract that increases protein stability, such as pharmacological saffron in S349L.
  • FIG. 3 shows the effect of mutations on protein stability and catalytic activity.
  • the intracellular PAH activity involved in determining the phenotype of individual mutant human PAHs is determined by the amount of protein and its catalytic activity.
  • FIG. 3 a line passing from the origin through the WT is drawn. This shows the true specific activity of wild-type human PAH and named it WT line. Assuming a mutation that only affects protein stability, it will move along the WT line. If the mutation affects both protein stability and catalytic activity, the mutation will appear below the WT line. The position will change depending on the influence of the mutation on each of the two, and the more severe the mutation, the closer to the origin.
  • R252Q, S349L, R408W are located below the WT line, which is consistent with the previously reported study results.
  • K42I is discussed below because it is attached to the WT line but similar to others belonging to regulatory domain mutations.
  • R252 typically appears to be a mutation that causes problems with catalytic activity and the results studied in other expression systems support this.
  • R252 is located in the catalytic domain and it is believed that alteration of this amino acid will disrupt stable interactions in the domain (Erlandsen H, et al. 2003 Pediatrics 112, 1557-1565).
  • R252Q recombinant protein obtained from E. coli and in vitro expression system showed 3 ⁇ 11.4% activity of wild type (Bj ⁇ rgo E, et al. 1998 Eur J Biochem 257, 1-10).
  • F39L, L48S, I65T and L255V are found on the WT line.
  • K42I is a mutation located on the WT line but with the same characteristics. This group means lower protein stability than wild type but higher catalytic activity. This is not common, but the rest is in the regulatory domain except for L255V.
  • regulatory domains are known to perform inhibitory functions by covering active sites (Fitzpatrick PF, 2012 Arch Biochem Biophys 519, 194201). Thus, mutations in the regulatory domain may exert an effect of increasing catalytic activity.
  • the cellular slime mold is a useful expression system capable of analyzing missense mutations at the protein and cellular levels in human PAH.
  • missense mutations in human PAH can be assessed quantitatively as their growth rate through expression in cellular slime molds, allowing for the characterization of mutant proteins for protein stability and catalytic activity under more in vivo conditions.
  • cell-based assays enable the study of structurally unstable mutant proteins.
  • Cellular slime molds are an economical research model and a stable expression system that will enable the detailed study of mutant proteins and furthermore provide an opportunity to analyze the pharmacological chaperone effects of tetrahydroterin and other candidate molecules. Will provide.
  • Cell-level mass spectrometry systems quantitative analysis using growth rates, especially for structurally unstable mutant PAH.
  • Protein level analysis system mutant PAHs can be studied without purification.
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Abstract

The present invention relates to a method for analyzing activity of human mutant phenylalanine hydroxylase (PAH) using cellular slime molds and, specifically, to a method for analyzing human mutant PAH activity by transforming a PAH gene, containing a phenylketonuria-related mutation, into cellular slime molds, followed by culture in the minimum essential medium lacking tyrosine, and then measuring the growth rate of the cellular slime molds.

Description

세포성점균을 이용한 인간 페닐알라닌 수산화효소의 활성분석 방법Activity Analysis Method of Human Phenylalanine Hydroxylase Using Cellular Bacteria
본 발명은 세포성점균을 이용한 인간 페닐알라닌 수산화효소(PAH)의 활성분석 방법에 관한 것으로, 구체적으로 페닐케톤뇨증 관련 돌연변이를 포함하는 PAH 유전자를 세포성점균에 형질전환 시키고, 티로신이 결핍된 최소영양배지에서 배양시킨 후, 세포성점균의 생장 속도를 측정함으로써 인간 돌연변이 PAH 활성을 분석하는 방법에 관한 것이다.The present invention relates to a method for analyzing the activity of human Phenylalanine Hydroxylase (PAH) using a cellular bacterium, specifically transforming a PAH gene containing a phenylketonuria-related mutation to a cellular bacterium, a minimal nutrient medium tyrosine deficient After culturing at, the present invention relates to a method for analyzing human mutant PAH activity by measuring the growth rate of cellular slime molds.
페닐알라닌 수산화효소(phenylalanine hydroxylase, PAH; EC 1.14.16.1)는 철이온(Fe2+)을 함유하는 효소로서 조효소인 테트라히드로비오테린(6R-L-erythro-tetrahydrobiopterin, BH4)과 분자산소(O2) 존재 하에서 L-페닐알라닌을 L-티로신으로 전환시키는 반응을 촉매한다. 인체에서 PAH는 간과 뇌 조직에 많이 존재하며, 간에서는 음식물로 섭취되는 페닐알라닌 대사에 주로 관여하며, 뇌에서는 도파민과 같은 신경전달물질의 합성에 중요하다 (Fitzpatrick PF. 2012 Arch Biochem Biophys 519, 194201). Phenylalanine hydroxylase (PAH; EC 1.14.16.1) is an enzyme containing iron ions (Fe 2+ ), coenzyme tetrahydrobioterin (6R-L-erythro-tetrahydrobiopterin (BH4)) and molecular oxygen (O 2). ) Catalyzes the conversion of L-phenylalanine to L-tyrosine in the presence of In humans, PAH is present in many liver and brain tissues, and is mainly involved in the metabolism of phenylalanine, which is consumed as a food in the liver, and is important for the synthesis of neurotransmitters such as dopamine in the brain (Fitzpatrick PF. 2012 Arch Biochem Biophys 519, 194201) .
PAH 유전자 돌연변이의 결과로 발생하는 효소활성의 결핍이나 유전자 발현의 저해로 인하여 열성유전질환인 페닐케톤뇨증 (phenylketonuria, PKU)이 야기된다. PKU는 간에서 페닐알라닌 대사가 원활히 이루어지지 않아 혈중 페닐케톤 화합물의 농도가 증가하여 생기는 질환으로 다양한 표현형으로 나타난다. PKU 환자들은 혈중 페닐알라닌 농도 600 μM을 기준으로 이보다 낮으면 고페닐알라닌증 (hyperphenylalaninemia, HPA), 높으면 PKU 그룹으로 구분된다. HPA는 다시 360 μM을 기준으로 치료가 필요 없는 그룹과 치료가 필요할 수도 있는 그룹으로 구분된다. PKU는 다시 mild PKU (600-900 μM), moderate PKU (900-1200 μM), classic PKU (>1200 μM)로 구분되며, 모두 강제적인 치료가 요구되고 있다 (Heintz C, et al. 2013 Hum Mutat 34, 927-936).Lack of enzymatic activity or inhibition of gene expression as a result of mutation of the PAH gene causes phenylketonuria (PKU), a recessive genetic disease. PKU is a disease caused by increased concentrations of phenylketone compounds in the blood due to poor metabolism of phenylalanine in the liver. PKU patients are divided into hyperphenylalaninemia (HPA) at lower than 600 μM in blood phenylalanine concentration and high PKU group. HPA is again divided into groups that do not need treatment and those that may need treatment based on 360 μM. PKU is divided into mild PKU (600-900 μM), moderate PKU (900-1200 μM), and classic PKU (> 1200 μM), all requiring compulsory treatment (Heintz C, et al. 2013 Hum Mutat 34, 927-936).
치료는 출생 초기부터 시작되며 그렇지 않을 경우 발달지체, 간질, 행동적인 문제, 우울증, 불안과 같은 신경정신 장애는 물론 심각한 지능저하가 발생한다. PKU 질환은 신생아 초기 단계에서 식이요법을 통해 혈중 페닐알라닌을 낮은 수준으로 유지하는 방법을 통해 증상을 완화시킬 수 있으나 평생 지속되어야 하는 것으로 알려져 있다. 그렇지만 식이요법에 대한 거부감이나 사회생활 때문에 엄격하게 지킬 수 없는 어려움이 따른다. 이와 같은 PKU 환자에게 PAH의 조효소인 BH4를 경구투여 함으로써 혈중 페닐알라닌을 낮출 수 있다는 것이 보고된 이 후, BH4는 PKU 환자를 위한 처방약으로 미국 FDA 승인을 받게 되었다 (Pey AL, et al. 2004 Hum Mutat 24, 388-399). BH4의 약리적 작용기작은 BH4가 L-Phe에 의한 효소의 활성화를 저해하기 때문이라고 알려져 있다 (‘pharmacological chaperone’이라 불림). 그러나 BH4는 모든 PKU 환자에서 효과적으로 작용하지 않기 때문에 BH4처럼 PAH 안정성을 증가시키는 pharmacological chaperone을 찾는 연구가 진행되고 있으며, 환자맞춤형 치료도 제안되고 있다 (Underhaug J, et al. 2012 Curr Top Med Chem 12, 2534-2545).Treatment begins early in life, or neuropsychiatric disorders such as developmental delays, epilepsy, behavioral problems, depression, and anxiety, as well as severe intelligence decline. PKU disease can be relieved by dietary low levels of phenylalanine in the early stages of newborn life, but it is known to last for life. However, refusal to diet or social life presents difficulties that cannot be strictly observed. Since it has been reported that oral administration of PAH coenzyme BH4 to PKU patients can lower blood phenylalanine, BH4 has been approved by the US FDA as a prescription drug for PKU patients (Pey AL, et al. 2004 Hum Mutat). 24, 388-399). The pharmacological mechanism of BH4 is known because BH4 inhibits the activation of enzymes by L-Phe (called 'pharmacological chaperone'). However, since BH4 does not work effectively in all PKU patients, studies are underway to find pharmacological chaperones that increase PAH stability like BH4, and patient-specific therapies have been proposed (Underhaug J, et al. 2012 Curr Top Med). Chem 12, 2534-2545).
PKU의 원인이 PAH 결핍에서 비롯된다는 사실은 1953년에 처음으로 보고되었다. 이어서 PAH의 유전자와 cDNA 서열이 확인되면서 PKU 환자들의 돌연변이 유전자 분석은 진단과 치료 차원에서 필수적으로 이루어지고 있다. 지금까지 확인된 PAH 유전자 돌연변이는 852종류로서 (BioPKU.org), 그 중 2/3 정도가 아미노산이 바뀐 미스센스(missense) 돌연변이에 해당된다. 이들 돌연변이의 표현형에 대한 연구는 단백질 차원에서 꾸준히 연구되어 왔다. 현재 다양한 돌연변이 PAH의 발현을 통해 연구된 결과는 PAHdb (http://www.pahdb.mcgill.ca/)에 정리되어 있다. In vitro에서의 단백질 분석은 주로 대장균, 효모, 포유동물 세포주, 또는 TNT-T7 망상적혈구 용해물(reticulocyte lysate)을 이용한 세포외 발현시스템을 이용하여 이루어졌다. 이러한 연구를 통해 PKU가 주로 단백질 접힘의 이상에서 생기는 질환(protein misfolding disease)임이 밝혀졌다 (Heintz C, et al. 2013 Hum Mutat 34, 927-936). 이는 미스센스 돌연변이 PAH의 경우, 단백질 접힘에 문제가 발생하여 단백질의 안정성이 감소하고 그로 인하여 단백질 분해가 촉진된다는 것이다. 이러한 원리에 입각하여 돌연변이가 PAH의 자연 상태의 안정성에 미치는 에너지 충격을 컴퓨터를 통해 분석하는 연구가 이루어지고 있다 (Shi Z1, et al. 2012 Proteins 80, 61-70). 그러나 이러한 노력에도 불구하고 유사한 PAH 유전형에 대한 in vivo와 in vitro 표현형 간에는 심각한 괴리가 발견되기도 한다 (Kayaalp E, et al. 1997 Am J Hum Genet 61, 1309-1317). 이는 부분적으로는 in vitro 발현시스템의 문제일 수도 있다. 이에, PKU를 유발하는 인간 PAH 유전자의 돌연변이를 정량적으로 단백질 수준에서 용이하게 분석할 수 있는 안정적인 시스템이 요구되는 실정이다.It was first reported in 1953 that the cause of PKU originated from PAH deficiency. Subsequently, the genes and cDNA sequences of PAH were identified, and mutant gene analysis of PKU patients is essential for diagnosis and treatment. The PAH gene mutations identified so far are 852 (BioPKU.org), about two-thirds of which are missense mutations with amino acid changes. Studies on the phenotype of these mutations have been steadily studied at the protein level. The results currently studied through the expression of various mutant PAHs are summarized in PAHdb (http://www.pahdb.mcgill.ca/). In vitro protein analysis was performed using an E. coli, yeast, mammalian cell line, or extracellular expression system using TNT-T7 reticulocyte lysate. These studies have shown that PKU is a protein misfolding disease mainly (Heintz C, et al. 2013 Hum Mutat 34, 927-936). This means that, in the case of missense mutant PAH, problems with protein folding occur, which reduces protein stability and thereby promotes protein degradation. Based on this principle, studies have been conducted to analyze the energy impact of mutations on the stability of PAH in its natural state (Shi Z1, et al. 2012 Proteins 80, 61-70). Despite these efforts, however, serious differences have been found between in vivo and in vitro phenotypes for similar PAH genotypes (Kayaalp E, et al. 1997 Am J Hum Genet 61, 1309-1317). This may be partly a problem of the in vitro expression system. Therefore, there is a need for a stable system capable of easily quantitatively analyzing the mutation of the human PAH gene causing PKU at the protein level.
따라서 본 발명의 목적은 세포수준의 분석 시스템으로서 페닐케톤뇨증 관련 돌연변이를 포함하는 PAH 유전자로 형질전환된 세포성점균의 생장 속도를 측정함으로써 인간 돌연변이 PAH 활성을 분석할 수 있는 방법을 제공하는데 있다.Accordingly, an object of the present invention is to provide a method for analyzing human mutant PAH activity by measuring the growth rate of a cellular bacterium transformed with a PAH gene including a phenylketonuria-related mutation as a cell-level analysis system.
본 발명의 다른 목적은, 상기 형질전환된 세포성점균을 이용한 페닐케톤뇨증 치료제의 스크리닝 방법을 제공하는데 있다.Another object of the present invention is to provide a method for screening a phenylketonuria drug using the transformed cellular slime mold.
본 발명의 한 양태에 따르면, 본 발명은, According to one aspect of the present invention,
a) 서열번호 1의 아미노산 서열로 표시되는 인간 PAH(phenylalanine hydroxylase)를 코딩하는 유전자에서 페닐케톤뇨증(phenylketonuria)을 야기시키는 돌연변이를 포함하는, 돌연변이 PAH 유전자를 세포성점균에 형질전환 시키는 단계; a) transforming a mutant PAH gene into a cellular bacterium, the mutation comprising a mutation causing phenylketonuria in a gene encoding human phenylalanine hydroxylase (PAH) represented by the amino acid sequence of SEQ ID NO: 1;
b) 상기 돌연변이 PAH 유전자로 치환된 세포성점균을 티로신(tyrosine)이 결핍된 최소영양배지에서 배양시키는 단계; 및 b) culturing the cellular slime mold substituted with the mutated PAH gene in a minimal nutrient medium lacking tyrosine; And
c) 상기 배양되는 세포성점균의 생장 속도를 측정하는 단계를 포함하는 세포성점균을 이용한 인간 돌연변이 PAH 활성분석 방법을 제공한다.c) it provides a method for assaying human mutant PAH activity using cellular bacteria comprising the step of measuring the growth rate of the cultured cellular bacteria.
본 발명에서는, 기존 대장균, 효모세포, 포유동물 세포주 및 인간세포를 비롯한 다양한 발현시스템을 이용한 PAH 활성 분석 시스템이 단백질의 안정성, in vivo와 in vitro 표현형 간의 심각한 차이 등으로 인해 분석과정에서의 신뢰성과 용이성의 개선점이 요구되는 점을 파악하고 이를 개선하기 위해, 세포성점균을 이용하여 인간 PAH 유전자 돌연변이의 표현형을 세포수준에서 분석할 수 있는 발현시스템을 개발하고자 하였다. 이를 위해 야생형을 비롯하여 페닐케톤뇨증을 야기시키는 것으로 알려진 다양한 돌연변이 각각을 가지는 인간 PAH cDNA를 발현벡터에 삽입하여 PAH 유전자가 치환된 세포성점균의 형질전환체에서 발현시키고 최소배지 (FM medium)에서 생장속도, PAH 효소활성, PAH 단백질량을 분석하였으며, 그 결과 서로 간에 밀접한 상관관계가 있음을 확인하였다. 따라서 본 발명의 결과는 세포성점균에서 PAH 효소 활성과 단백질 안정성을 반영하는 세포증식 속도의 차이를 통해 인간 PAH 유전자 돌연변이의 표현형을 용이하게 분석할 수 있음을 제안한다.In the present invention, the PAH activity analysis system using various expression systems, including E. coli, yeast cells, mammalian cell lines and human cells, has a high reliability and reliability in the analysis process due to protein stability, serious differences between in vivo and in vitro phenotype. In order to identify and improve the need for improvement of ease, we tried to develop an expression system that can analyze the phenotype of human PAH gene mutations at the cellular level using cellular bacteria. To this end, human PAH cDNA containing each of the various mutations known to cause phenylketonuria, including wild type, was inserted into an expression vector, expressed in a transformant of the PAH gene-substituted cellular slime mold, and grown at the minimum medium (FM medium). , PAH enzyme activity, and the amount of PAH protein were analyzed. As a result, it was confirmed that there is a close correlation between each other. Therefore, the results of the present invention suggest that the phenotype of human PAH gene mutations can be easily analyzed through the difference in cell proliferation rate reflecting PAH enzyme activity and protein stability in cellular bacteria.
한편, 상기 세포성점균은 인간세포에 비해 매우 높은 수준의 L-erythro-tetrahydrobiopterin (BH4)와 그 이성질체인 D-threo-BH4 (DH4)를 생산한다 (두 가지를 합쳐서 테트라히드로테린이라 부름). 이들은 PAH의 조효소이자 단백질 안정성에 기여하는 소위 pharmacological chaperone으로 작용한다. 따라서 세포성점균은 세포외에서 이들을 첨가하지 않고도 이들에 의한 pharmacological chaperone 효과를 발휘한다. 이를 검증하기 위하여 테트라히드로테린 생합성에 관여하는 세피아테린 환원효소(sepiapterin reductase) 유전자가 치환된 돌연변이 세포성점균 (spr -)에서도 돌연변이 PAH를 발현시켰다. 그 결과 PAH 유전자에 결함을 가진 페닐케톤뇨증 환자들 중에서 BH4 약물에 반응하는 돌연변이로 알려진 F39L, K42I, I65T의 경우 PAH 단백질량의 감소를 확인하였다. 이는 세포성점균을 통해 BH4의 pharmacological chaperone 효과를 검증할 수 있음을 보여주는 동시에 다른 pharmacological chaperone의 개발에도 이용할 수 있음을 제시한다. 그 가능성을 타진하기 위하여, 형질전환된 세포성점균을 최소배지 대신에 천연성분들의 집합체인 효모추출물(yeast extract)이 포함된 영양배지(HL5 medium)에서 배양하였다. 그 결과 8가지 돌연변이 PAH 중에서 S349L과 R408W의 경우에만 단백질량의 증가와 더불어 활성증가도 관찰되었다. 아직 영양배지의 어떤 성분이 효과를 발휘하는지는 확인되지 않았다. 그러나 이 연구결과의 중요성은, 본 발명의 분석시스템을 이용하여 S349L과 R408W와 같이 단백질구조에 심각한 영향을 주는 돌연변이에 대한 연구가 가능하다는 것이다.On the other hand, the cellular slime mold produces very high levels of L-erythro-tetrahydrobiopterin (BH4) and its isomer D-threo-BH4 (DH4) compared to human cells (the two are called tetrahydroterin). They act as a coenzyme of PAH and as a so-called pharmacological chaperone that contributes to protein stability. Therefore, cellular slime molds exert their pharmacological chaperone effect without adding them extracellularly. In order to verify this, tetrahydro sepia aminopterin reductase (sepiapterin reductase) gene is mutated cells Slime Molds substituted (spr -) involved in the biosynthesis of aminopterin was expressed in the mutant PAH. As a result, the decrease in the amount of PAH protein was confirmed in the case of F39L, K42I, and I65T, which are known to be mutations in response to the BH4 drug among patients with phenylketonuria having a defect in the PAH gene. This demonstrates that the cellular slime mold can verify the pharmacological chaperone effect of BH4 and that it can be used to develop other pharmacological chaperones. To explore the possibility, transformed cellular slime molds were cultured in nutrient medium (HL5 medium) containing yeast extract, a collection of natural ingredients, instead of minimal medium. As a result, only S349L and R408W of 8 mutant PAHs showed increased protein and increased activity. It is not yet known which component of the nutrient medium will work. However, the significance of these findings is that, using the assay system of the present invention, it is possible to study mutations that seriously affect protein structures such as S349L and R408W.
지금까지 세포성점균을 인간 돌연변이 PAH 발현에 사용한 연구 보고는 없다. 따라서 본 발명을 통해 얻어진 결과를 다양한 발현시스템을 통해 이미 발표된 연구 결과들과 비교분석 하였다. 그 결과 L255V를 제외한 나머지 7가지 돌연변이 단백질들의 단백질 안정성과 효소 활성에 대한 분석결과가 매우 유사함을 보여주었다. 이는 세포성점균이 인간 PAH와 관련된 연구에 적합하다는 것을 증명하는 것이다. To date, there have been no reports on the use of cellular slime molds for expression of human mutant PAH. Therefore, the results obtained through the present invention were compared with the results of researches already published through various expression systems. As a result, the analysis of protein stability and enzyme activity of seven mutant proteins except L255V showed very similar results. This proves that cellular slime molds are suitable for studies involving human PAH.
본 발명에서, 상기 ‘세포성점균(cellular slime mold)’은 균류로서 진핵균아계 세포점균문의 한 강에 속하며, 단핵인 아메바상 세포가 모여서 접합변형체를 만들며 나중에 누적자실체를 형성한다. 모인 세포의 하나하나가 전생활사를 통하여 완전히 독립되어 존재하며, 곰팡이류보다 오히려 원생동물인 아메바에 가까운 것으로 여겨진다. 세포성점균에 속하는 것 중 딕티오스텔륨 디스코이뎀(Dictyiostelium discoidem)은 그 생활사가 잘 밝혀진 종이다. 또한 상기 세포성점균은 생의학 분야의 연구에 중요하게 사용되는 연구모델 생물체로서 진핵생물의 단백질 발현에 유용한 것으로 알려져 있다 (Arya R, et al. 2008 FASEB J 22, 4055-4066). 상기 세포성점균의 PAH는, 인간 PAH와 매우 유사한 구조와 아미노산 서열을 가진다. 인간 PAH와 마찬가지로 동형사량체 효소로서 각각의 단위체는 3개의 도메인(아미노 말단의 조절 도메인; 기질과 조효소가 결합하는 촉매 도메인; 및 카르복시 말단의 다량체 도메인)으로 구분된다. 흥미롭게도 세포성점균은 BH4 외에도 그 이성질체인 D-threo-isomer (DH4)를 다량 생산하며 (Kim HL, et al. 2012 FEBS Lett 586, 3596-3600), 이들은 (테트라히드로테린이 불림) 모두 인간과 세포성점균의 PAH에 조효소로 작용한다 (Siltberg-Liberles J, et al. 2008 Gene 427, 8692). In the present invention, the 'cellular slime mold' is a fungi belonging to a river of eukaryotic subtype cell slime molds, mononuclear amoeba-like cells are collected to form a junctional variant and later form an accumulation body. Each of the collected cells is completely independent of the whole life history, and is thought to be closer to the protozoa, amoeba, rather than fungi. Dictyiostelium discoidem , one of the cellular bacteria , is a species whose life history is well known. In addition, the cell slime mold is known to be useful for eukaryotic protein expression as a research model organism that is important for biomedical research (Arya R, et al. 2008 FASEB J 22, 4055-4066). The PAH of the cellular bacilli has a structure and amino acid sequence very similar to that of human PAH. As with human PAH, each homomer as an isomeric tetramer enzyme is divided into three domains: an amino terminal regulatory domain; a catalytic domain to which a substrate and a coenzyme bind; and a carboxy terminal multimeric domain. Interestingly, the cellular bacillus produces a large amount of the isomer D-threo-isomer (DH4) in addition to BH4 (Kim HL, et al. 2012 FEBS Lett 586, 3596-3600), both of which are called tetrahydroterins, act as coenzymes for PAH in human and cellular bacteria (Siltberg-Liberles J, et al. 2008 Gene 427, 8692).
본 발명의 PAH 활성분석 방법에 있어서, 상기 인간 PAH를 코딩하는 유전자는 인간 페닐알라닌 수산화효소(hPAH) 단백질을 암호화는 어떠한 염기서열을 가질 수 있으나, 바람직하게는 서열번호 1의 아미노산 서열을 코딩하는 염기서열이며, 더욱 바람직하게는 서열번호 2의 염기서열을 갖는 hPAH cDNA(ORF)일 수 있다. In the PAH activity analysis method of the present invention, the gene encoding the human PAH may have any base sequence encoding a human phenylalanine hydroxylase (hPAH) protein, but preferably a base encoding the amino acid sequence of SEQ ID NO: 1 Sequence, and more preferably hPAH cDNA (ORF) having a nucleotide sequence of SEQ ID NO: 2.
본 발명의 PAH 활성분석 방법에 있어서, 상기 “페닐케톤뇨증을 야기시키는 돌연변이”는 PAH 단백질을 코딩하는 유전자에서 염기의 치환, 삭제, 삽입 등 뉴클레오티드의 변화에 따른 효소 활성의 결핍이나 유전자 발현 저해로 인해 페닐케톤뇨증을 유발하는 모든 돌연변이를 포함한다. 본 발명은 이러한 돌연변이 PAH의 활성을 분석하는데 세포성점균을 이용한다는 점에 기술적 특징이 있으므로, 세포성점균의 형질전환에 이용되는 돌연변이 PAH는 특별히 제한될 이유가 없다. 지금까지 확인된 PAH 유전자 돌연변이는 852종류로서 (BioPKU.org), 그 중 2/3 정도가 아미노산이 바뀐 미스센스(missense) 돌연변이에 해당되며, 현재 다양한 돌연변이 PAH의 발현을 통해 연구된 결과는 PAHdb (http://www.pahdb.mcgill.ca/)에 정리되어 있다. In the PAH activity analysis method of the present invention, the “mutation causing phenylketonuria” is due to a lack of enzymatic activity or gene expression inhibition due to nucleotide changes such as substitution, deletion and insertion of bases in a gene encoding PAH protein. All mutations that cause phenylketonuria are included. Since the present invention has a technical feature in that the cellular slime mold is used to analyze the activity of such mutant PAH, the mutant PAH used for transforming the cellular slime mold is not particularly limited. The PAH gene mutations identified so far are 852 (BioPKU.org), two-thirds of which are missense mutations with amino acid changes. (http://www.pahdb.mcgill.ca/).
본 발명의 일실시예에서, 상기 돌연변이는 서열번호 1로 표시되는 인간 PAH의 아미노산 서열에서 아미노산 위치 F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L 및 R408W로 구성된 군에서 선택되는 미스센스 돌연변이(missense mutation)이다.In one embodiment of the invention, the mutation is a missense selected from the group consisting of amino acid positions F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W in the amino acid sequence of human PAH represented by SEQ ID NO: 1 It's a mutation.
본 발명에서 용어 ‘미스센스 돌연변이’는 DNA의 염기 서열 중 한 개의 염기가 다른 염기로 치환되어 아미노산의 코돈이 다른 코돈으로 바뀌는 결과를 초래하는 돌연변이의 종류로서, ‘자리지정 돌연변이 또는 점 돌연변이(site-directed mutagenesis)’를 통해 이루어질 수 있다. In the present invention, the term 'missense mutation' refers to a type of mutation that causes one base of the DNA to be replaced with another base to change the codon of an amino acid to another codon. -directed mutagenesis).
본 발명에서, 인간 PAH 단백질을 코딩하는 유전자 서열은 본 발명에서 선택된 돌연변이 생성을 위한 시작점으로 이용된다. 당업자라면 상기 열거된 아미노산 위치들의 돌연변이 생성을 위하여 다양한 공지의 표준적인 점 돌연변이 유발(site-directed mutagenesis) 방법을 임의로 사용할 수 있다 (Sambrook, J. et al. (1989), supra). 흔히 사용되는 방법의 하나는 합성 올리고뉴클레오티드 혼합물을 사용한 PCR을 이용하여 돌연변이들을 도입시키는 것이다.In the present invention, the gene sequence encoding human PAH protein is used as a starting point for the generation of mutations selected in the present invention. One skilled in the art can optionally use various known standard site-directed mutagenesis methods for mutagenesis of the amino acid positions listed above (Sambrook, J. et al. (1989), supra). One commonly used method is to introduce mutations using PCR using a synthetic oligonucleotide mixture.
본 발명의 일실시예에서, 상기 미스센스 돌연변이는 서열번호 3 내지 18로 표시되는 프라이머를 이용하여 해당 코돈의 염기를 치환함으로써 수행될 수 있다. In one embodiment of the present invention, the missense mutation can be carried out by replacing the base of the codon using a primer represented by SEQ ID NO: 3 to 18.
이러한 돌연변이가 유발된 PAH 유전자는 적절한 발현 벡터에 삽입되어 숙주세포인 세포성점균에 형질전환될 수 있다. PAH gene in which such a mutation is induced can be inserted into an appropriate expression vector and transformed into a host cell, a cellular bacterium.
본 발명에서 용어, ‘벡터’는 연결되어 있는 다른 핵산을 운반할 수 있는 핵산 분자를 의미하며, 벡터의 하나의 유형인 ‘플라스미드’는 그 안에 추가적으로 DNA 조각을 연결시킬 수 있는 환형의 이중 가닥 DNA 루프를 의미한다.In the present invention, the term 'vector' refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked, and one type of vector, 'plasmid', is a circular double-stranded DNA that can additionally connect DNA fragments therein. It means a loop.
본 발명에서 용어, ‘발현벡터’는 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 재조합 벡터로서, 작동 가능하도록 연결된 목적 단백질을 코딩하는 유전자의 발현을 지시하는 벡터를 의미한다. 상기 발현벡터는 이에 제한되지는 않으나 플라스미드 벡터, 코즈미드 벡터, 박테리오파지 벡터 및 아데노바이러스 벡터, 레트로바이러스 벡터와 같은 바이러스 벡터 등을 포함하며, 재조합 DNA 기술의 사용에 있어서 바람직하게는 플라스미드 벡터일 수 있다.As used herein, the term 'expression vector' refers to a vector that directs the expression of a gene encoding a target protein that is operably linked as a recombinant vector capable of expressing a target protein in a suitable host cell. The expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector such as an adenovirus vector, a retroviral vector, and the like, and preferably in the use of recombinant DNA technology. .
본 발명에서 용어, ‘형질전환’은 DNA를 숙주로 도입하여 DNA가 염색체의 인자로서 또는 염색체 통합 완성에 의해 복제 가능하게 되는 것으로 외부의 DNA를 세포 내로 도입하여 인위적으로 유전적인 변화를 일으키는 현상을 의미한다. 본 발명의 숙주세포는 세포성점균이며, 본 발명의 일실시예에서는 딕티오스텔리움 디스코이데움(Dictyostelium discoideum)을 세포성점균의 대표적인 예로 사용하였다.In the present invention, the term 'transformation' refers to a phenomenon in which DNA is introduced into a host so that DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration, thereby introducing an external DNA into a cell and causing an artificial genetic change. it means. The host cell of the present invention is a cellular bacteria, and in one embodiment of the present invention, Dictyostelium discoideum was used as a representative example of the cellular bacteria.
본 발명의 형질전환 방법은 임의의 형질전환 방법이 사용될 수 있으며, 당업계의 통상적인 방법에 따라 용이하게 수행할 수 있다. 일반적으로 형질전환 방법에는 CaCl2 침전법, CaCl2 방법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 매개된 형질전환 방법 등이 있다. 따라서, 본 발명에서 상기 형질전환 방법을 제한 없이 이용하여 본 발명의 돌연변이 PAH 유전자를 포함하는 발현벡터를 세포성점균으로 도입함으로써, 형질전환체를 획득할 수 있다.As the transformation method of the present invention, any transformation method may be used, and may be easily performed according to conventional methods in the art. In general, the Hanahan method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, and silicon carbide, which have improved efficiency by using a CaCl 2 precipitation method and a reducing material called DMSO (dimethyl sulfoxide) in the CaCl 2 method Agitation with fibers, agrobacterial mediated transformation, transformation with PEG, dextran sulfate, lipofectamine mediated transformation, and the like. Therefore, the transformant can be obtained by introducing the expression vector including the mutated PAH gene of the present invention into a cellular bacterium using the transformation method without limitation in the present invention.
본 발명의 PAH 활성분석 방법에 있어서, 상기 세포성점균의 생장 속도는 PAH 활성과 선형상관관계에 있는 것을 특징으로 한다.In the PAH activity analysis method of the present invention, the growth rate of the cellular mycobacteria is characterized by a linear correlation with PAH activity.
본 발명의 바람직한 구현예에서, PAH 유전자가 치환된 돌연변이 세포성점균(pah -), 야생형 세포성점균(WT) 및 본 발명의 돌연변이 인간 PAH 유전자 각각이 삽입된 발현벡터를 넣은 형질전환체를 이용하여 생장속도와 PAH 활성 간의 상관관계를 확인한 결과, 도 1의 A에 나타난 바와 같이, 형질전환체 마다 생장속도와 PAH 활성의 수준은 상관관계를 나타내었다. 이러한 세포성 점균의 생장속도와 PAH 활성 간에 밀접한 상관관계가 있음을 확인하기 위해 선형회귀분석을 실시한 결과, 도 1의 B에 나타난 바와 같이, 통계적으로 유의성이 있는 선형상관관계를 확인하였다. 이러한 결과는 본 발명의 세포성점균을 이용한 PAH 활성분석 방법으로부터 pah -에서 상보적 발현을 통해 그 생장속도로서 미스센스 돌연변이를 가진 인간 PAH를 정량적으로 평가할 수 있음을 의미한다. 생장속도는 PAH 활성보다 측정하기가 용이하다는 점에서 본 발명은 이용가치가 매우 높은 기술로 판단된다. In a preferred embodiment of the present invention, a transformant containing a mutant cytoplasmic bacterium ( pah − ) substituted with a PAH gene, a wild-type cellular bacterial (WT), and an expression vector into which a mutant human PAH gene of the present invention is inserted, respectively As a result of confirming the correlation between the growth rate and PAH activity, as shown in A of FIG. 1, the growth rate and the level of PAH activity for each transformant showed a correlation. As a result of performing a linear regression analysis to confirm that there is a close correlation between the growth rate of the cellular slime mold and PAH activity, as shown in B of FIG. 1, a statistically significant linear correlation was confirmed. These results indicate that quantitative evaluation of human PAH with missense mutations as its growth rate is possible through complementary expression at pah − from the PAH activity assay method using the cellular bacterium of the present invention. As the growth rate is easier to measure than PAH activity, the present invention is judged to be a very high value technology.
본 발명의 다른 양태에 따르면, 본 발명은, According to another aspect of the present invention, the present invention,
1) 서열번호 1의 아미노산 서열로 표시되는 인간 PAH(phenylalanine hydroxylase)에서 F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L 및 R408W로 구성된 군에서 선택된 아미노산이 치환된 돌연변이 hPAH를 코딩하는 폴리뉴클레오티드가 도입된 재조합벡터로 형질전환된 세포성점균을 제조하는 단계; 1) A poly encoding a mutant hPAH substituted with an amino acid selected from the group consisting of F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W in human phenylalanine hydroxylase represented by the amino acid sequence of SEQ ID NO: 1 Preparing a cellular bacterium transformed with the recombinant vector into which the nucleotide is introduced;
2) 상기 형질전환체된 세포성점균을 PAH에 대한 약리학적 샤프론(pharmacological chaperone) 후보물질이 첨가된 티로신-결핍 최소배양배지에서 배양하는 단계; 및 2) culturing the transformed cellular mycobacteria in a tyrosine-deficient minimal culture medium to which a pharmacological chaperone candidate for PAH has been added; And
3) 상기 배양 후 세포성점균의 생장 속도가 후보물질 비처리군과 비교하여 증가된 경우, 상기 후보물질을 PAH에 대한 약리학적 샤프론으로 판단하는 단계를 포함하는, 세포성점균을 이용한 페닐케톤뇨증(phenylketonuria) 치료제의 스크리닝 방법을 제공한다.3) when the growth rate of the cellular bacterium after the culture is increased compared to the non-treated group of the candidate substance, the candidate substance is determined as pharmacological saffron for PAH, phenylketonuria using the cellular bacterium ( phenylketonuria) provides a method for screening a therapeutic agent.
본 발명의 스크리닝 방법에 있어서, 상기 1) 단계에서 재조합벡터는 돌연변이 hPAH 유전자가 pDXA-3H 벡터에 삽입된 재조합벡터로서, 도 5에 개시된 개열지도를 갖는 재조합벡터이다. In the screening method of the present invention, in the step 1), the recombinant vector is a recombinant vector having a mutated hPAH gene inserted into a pDXA-3H vector and has a cleavage map as shown in FIG. 5.
본 발명에서, 상기 “약리학적 샤프론(pharmacological chaperone)”은 페닐케톤뇨증(PKU)이 주로 단백질 접힘의 이상에서 생기는 질환(protein misfolding disease)으로 밝혀져 있고 (Heintz C, et al. 2013 Hum Mutat 34, 927-936), 특히 미스센스 돌연변이 PAH의 경우, 단백질 접힘에 문제가 발생하여 단백질의 안정성이 감소하고 그로 인하여 단백질 분해가 촉진되기 때문에, 단백질 접힘을 도와주는 역할을 하거나 잘못 접힌(misfolded) 단백질들을 수정하고 기능을 회복하는데 도움을 주는 물질 또는 소분자를 의미하는 것으로 해석된다. 예들 들어, 대한민국 공개특허 제10-2007-0005550호에서는, PKU의 치료를 위해 BH4와 같은 물질을 투여하는 방법에 대해 개시하고 있다. 그러나 BH4는 모든 PKU 환자에서 효과적으로 작용하지 않기 때문에 BH4처럼 PAH 안정성을 증가시키는 약리학적 샤프론을 찾는 연구가 진행되고 있으며, 환자맞춤형 치료도 제안되고 있다.In the present invention, the “pharmacological chaperone” has been found to be a protein misfolding disease in which phenylketonuria (PKU) is mainly caused by abnormalities in protein folding (Heintz C, et al. 2013 Hum Mutat 34, 927 936), especially in the case of missense mutant PAHs, which contribute to protein folding or correct misfolded proteins because of the problem of protein folding, which reduces protein stability and thereby promotes protein degradation. And substances or small molecules that help to restore function. For example, Korean Patent Publication No. 10-2007-0005550 discloses a method of administering a substance such as BH4 for the treatment of PKU. However, since BH4 does not work effectively in all PKU patients, studies are underway to find pharmacological chaperones that increase PAH stability like BH4.
본 발명의 스크리닝 방법에 있어서, 상기 2) 단계에서 약리학적 샤프론 후보물질은 천연화합물, 합성화합물, 효소, 단백질 또는 핵산일 수 있다. 상기 후보물질이 첨가된 배지에서 배양되는 세포성점균의 생장 속도를 측정하여 상기 후보물질이 돌연변이 PAH의 활성에 어떠한 영향을 미치는지 관찰함으로써, 상기 후보물질을 PAH에 대한 약리학적 샤프론으로 판단할 수 있다. 다시 말해서, 상기 첨가된 후보물질에 의해 PAH 단백질량 또는 PAH 활성이 회복된다면, 상기 후보물질은 돌연변이 PAH에 대한 약학적 샤프론과 유사한 역할을 하는 것으로 기대할 수 있다. 그러한 경우 상기 후보물질은 돌연변이 PAH의 단백질 안정성을 향상시키는 것으로 기대할 수 있다.In the screening method of the present invention, the pharmacological chaperone candidate in step 2) may be a natural compound, a synthetic compound, an enzyme, a protein or a nucleic acid. By measuring the growth rate of the cellular mycobacteria cultured in the medium to which the candidate is added, the candidate substance can be judged as a pharmacological saffron for PAH by observing how the candidate substance affects the activity of the mutant PAH. . In other words, if the amount of PAH protein or PAH activity is restored by the added candidate, the candidate can be expected to play a similar role as the pharmaceutical chaperone for mutant PAH. In such cases the candidates can be expected to improve the protein stability of the mutant PAH.
본 발명은 다음과 같은 효과 및 이점을 가진다.The present invention has the following effects and advantages.
1. 돌연변이 PAH 연구시스템으로서의 세포성점균의 적합성 검증: 8가지 돌연변이 단백질들의 활성과 단백질량 분석을 통해 인간세포를 비롯한 다양한 발현시스템에서 연구된 결과와 유사한 결과 도출.1. Verification of the cellular bacillus as a mutant PAH research system: Activity and protein content analysis of 8 mutant proteins yielded results similar to those studied in various expression systems including human cells.
2. 세포수준의 대량분석 시스템: 생장속도를 이용한 정량적 분석이 가능하며, 특히 구조적으로 불안정한 돌연변이 PAH에 대한 연구 가능.2. Cell-level mass spectrometry systems: quantitative analysis using growth rates, especially for structurally unstable mutant PAH.
3. 단백질 수준의 분석 시스템: 정제과정 없이도 돌연변이 PAH에 대한 연구 가능. 3. Protein level analysis system: mutant PAHs can be studied without purification.
4. 경제적이고 안정적인 발현시스템: 개개의 PKU 돌연변이 단백질에 대한 심화 연구 가능. 세포성점균은 대장균과 유사한 배지를 사용하므로 포유동물세포 배양에 비해 저렴.4. Economical and stable expression system: Further research on individual PKU mutant proteins is possible. Cellular slime is cheaper than mammalian cell culture because it uses a medium similar to E. coli.
5. 환자 맞춤 치료에 이용: PKU 환자의 돌연변이 유전형에 집중된 연구 가능.5. Use in patient-specific treatment: focused research on mutant genotypes in PKU patients.
도 1은 돌연변이 hPAH cDNA로 형질전환된 pah - 균주의 생장속도 및 PAH 활성의 비교 분석 결과이다. (A) 생장속도 및 PAH 활성. 생장속도는 야생형(WT)의 퍼센트로 나타내었다. (B) PAH 활성과 생장속도 사이의 정량적 상관관계. 시그마 플롯(sigma plot)이 데이터 분석에 사용되었다. 상관계수(r2)는 95% 신뢰구간으로 제공된다 (dotted lines). 1 is a comparative analysis of the growth rate and PAH activity of pah - strain transformed with mutant hPAH cDNA. (A) growth rate and PAH activity. Growth rate is expressed as a percentage of wild type (WT). (B) Quantitative correlation between PAH activity and growth rate. Sigma plots were used for data analysis. The correlation coefficient (r2) is given as 95% confidence intervals (dotted lines).
도 2는 hPAH에 대한 세포내 테트라히드로테린(tetrahydropteridine) 수준 및 영양배지에 대한 효과를 나타낸 결과이다. (A) 화학발광 반응으로 분석한 웨스턴 블롯의 결과: I, FM 배지에서 배양된 pah - 세포; II, FM 배지에서 배양된 spr - 세포; III, HL5 배지에서 배양된 pah - 세포. 총 단백질 50 μg과 동량의 조추출물이 12.5% SDS-PAGE 및 웨스턴 블로팅으로 분석되었다. (B) pah - 및 spr - 세포에서 PAH 잔류 단백질의 양. (C) FM 및 HL5 배지에서 배양된 pah - 세포에서 잔류 PAH 단백질의 양. (D) FM 및 HL5 배지에서 배양된 pah - 세포로부터 측정된 PAH 활성. 모든 데이터는 야생형(WT)의 퍼센트로 나타내었다. Figure 2 is a result showing the effect on intracellular tetrahydropteridine levels and nutrient medium on hPAH. (A) Results of Western blot analyzed by chemiluminescent reaction: I, pah - cells cultured in FM medium; II, spr - cells cultured in FM medium; III, pah - cells cultured in HL5 medium. 50 μg of total protein and the same crude extract were analyzed by 12.5% SDS-PAGE and Western blotting. (B) The amount of PAH residual protein in pah - and spr - cells. (C) Amount of residual PAH protein in pah - cells cultured in FM and HL5 medium. (D) PAH activity measured from pah - cells cultured in FM and HL5 medium. All data are expressed as percentage of wild type (WT).
도 3은 pah - 균주에서 발현된 hPAHs의 PAH 활성 및 단백질 수준 사이의 관계를 나타낸 것이다. 도 1의 B와 도 2에 있는 평균값 데이터가 야생형의 퍼센트로 표시되었다. 3 shows the relationship between PAH activity and protein levels of hPAHs expressed in the pah − strain. The mean data in FIG. 1B and FIG. 2 are expressed as percentage of wild type.
도 4는 S349L 균주를 효모추출물이 첨가된 FM 배지에서 키운 후 웨스턴 블롯으로 분석한 결과이다. 4 is a result of Western Blot analysis after growing the S349L strain in FM medium to which yeast extract is added.
도 5는 본 발명에 따른 돌연변이 hPAH 유전자가 도입된 재조합벡터의 개열지도를 나타낸 것이다. Figure 5 shows a cleavage map of the recombinant vector into which the mutant hPAH gene was introduced according to the present invention.
도 6은 인간 PAH의 아미노산 서열 및 치환될 아미노산 위치를 표시한 것이다. 6 shows the amino acid sequence of human PAH and the amino acid position to be substituted.
이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예 1. 균주 배양Example 1. Strain Culture
야생형 세포성점균(Dictyostelium discoideum Ax2: Dictybase (http://dictybase.org/) Stock center에서 구입)은 HL5 배지 (리터당 10 g glucose, 5 g yeast extract, 10 g protease peptone, 0.35 g KH2PO4, 0.35g Na2HPO4·12H2O, pH6.4)에 100 μg/ml의 스트렙토마이신(streptomycin sulfate)과 100 U/ml의 페니실린(benzylpenicillin potassium)을 첨가하여 배양하였다 (Watts DJ et al. 1970 Biochem J 119, 171174). 이전 연구(Kim HL, et al. 2012, FEBS Lett. 586: 3596-3600)를 통해 보유하고 있던 PAH 녹아웃 돌연변이체(pah -)와 세피아테린 환원효소 넉아웃 돌연변이체 (spr -)는 HL5 배지에 10 μg/ml의 Blasticidine S를 첨가하여 배양하였다. 인간 PAH cDNA로 형질전환시킨 돌연변이체들은 10 μg/ml의 Blasticidine S와 G418 각각이 첨가된 배지에서 유지되었다. 상기 pah -와 pr -에는 Blasticidine S에 대한 내성유전자가 재조합을 통해 염색체에 삽입되어 있고, hPAH-pDXA-3H 발현벡터에는 G418에 대한 내성유전자가 삽입되어 있다. 돌연변이체들은 FM 최소배양배지(ForMediumTM, UK)에서도 배양되었다. Wild-type Cytomegalovirus ( Dictyostelium) discoideum Ax2: Dictybase (purchased from http://dictybase.org/ ) stock center is HL5 medium (10 g glucose per liter, 5 g yeast extract, 10 g protease peptone, 0.35 g KH 2 PO 4 , 0.35 g Na 2 HPO Incubated with 100 μg / ml of streptomycin sulfate and 100 U / ml of penzylpenicillin potassium to 4 12 H 2 O, pH6.4 (Watts DJ et al. 1970 Biochem J 119, 171174). ). Previous study (Kim HL, et al. 2012, FEBS Lett . 586: 3596-3600), the PAH knockout mutant (pah which has through-) and sepia aminopterin reductase knockout mutant (spr -) was cultured by the addition of Blasticidine S of 10 μg / ml in HL5 medium. Mutants transformed with human PAH cDNA were maintained in medium supplemented with 10 μg / ml Blasticidine S and G418 respectively. The pah - and pr - has a resistance gene for G418-resistant gene is inserted into the chromosome via recombination, hPAH-pDXA-3H expression vectors for Blasticidine S is inserted. Mutants were also cultured in FM minimal culture medium (ForMedium ™ , UK).
실시예Example 2. 자리지정 돌연변이 2. Placement mutation
서열번호 1로 표시되는 인간 PAH의 아미노산 서열상으로 39, 42, 48, 65, 252, 255, 349, 408에 위치한 아미노산 잔기를 대체하기 위하여 overlap extension PCR 방법을 사용하였다 (Heckman KL et al., 2007 Nat Protocols 2, 924-932). 각각에 해당하는 치환시킬 염기서열을 포함하는 상보적인 두 개의 PCR 프라이머를 하기 표 1과 같이 제조하였다.The overlap extension PCR method was used to replace amino acid residues located at 39, 42, 48, 65, 252, 255, 349, 408 on the amino acid sequence of human PAH represented by SEQ ID NO: 1 (Heckman KL et al., 2007 Nat Protocols 2, 924-932). Two complementary PCR primers, each containing a base sequence to be substituted, were prepared as shown in Table 1 below.
돌연변이 위치Mutation location 프라이머 서열Primer sequence 서열번호SEQ ID NO:
F39LF39L 5’-CCATATCACTGATCTT G TCACTCAAAGAAGAA-3’5'-CCATATCACTGATCTT G TCACTCAAAGAAGAA-3 ' 33
5’-TTCTTCTTTGAGTGA C AAGATCAGTGATATGG-3’5'-TTCTTCTTTGAGTGA C AAGATCAGTGATATGG-3 ' 44
K42IK42I 5’-CTGATCTTCTCACTCA T AGAAGAAGTTGGTGC-3’5'-CTGATCTTCTCACTCA T AGAAGAAGTTGGTGC-3 ' 55
5’-GCACCAACTTCTTCT A TGAGTGAGAAGATCAG-3’5'-GCACCAACTTCTTCT A TGAGTGAGAAGATCAG-3 ' 66
L48SL48S 5’-GAAGTTGGTGCAT C GGCCAAAGTATT-3’5'-GAAGTTGGTGCAT C GGCCAAAGTATT-3 ' 77
5’-AATACTTTGGCC G ATGCACCAACTTC-3’5'-AATACTTTGGCC G ATGCACCAACTTC-3 ' 88
I65TI65T 5’-GTAAACCTGACCCACA C TGAATCTAGACCTTC-3’5'-GTAAACCTGACCCACA C TGAATCTAGACCTTC-3 ' 99
5’-GAAGGTCTAGATTCA G TGTGGGTCAGGTTTAC-3’5'-GAAGGTCTAGATTCA G TGTGGGTCAGGTTTAC-3 ' 1010
R252QR252Q 5’-TGCTTTCCTCTC A GGATTTCTTGGGTG-3’5'-TGCTTTCCTCTC A GGATTTCTTGGGTG-3 ' 1111
5’-CACCCAAGAAATCC T GAGAGGAAAGCA-3’5'-CACCCAAGAAATCC T GAGAGGAAAGCA-3 ' 1212
L255VL255V 5’-CTCGGGATTTC G TGGGTGGCCTG-3’5'-CTCGGGATTTC G TGGGTGGCCTG-3 ' 1313
5’-CAGGCCACCCA C GAAATCCCGAG-3’5'-CAGGCCACCCA C GAAATCCCGAG-3 ' 1414
S349LS349L 5’-CTGGGCTCCTGT T ATCCTTTGGTGAATT-3’5'-CTGGGCTCCTGT T ATCCTTTGGTGAATT-3 ' 1515
5’-AATTCACCAAAGGAT A ACAGGAGCCCAG-3’5'-AATTCACCAAAGGAT A ACAGGAGCCCAG-3 ' 1616
R408WR408W 5’-GCCACAATACCT T GGCCCTTCTCAG-3’5'-GCCACAATACCT T GGCCCTTCTCAG-3 ' 1717
5’-CTGAGAAGGGCC A AGGTATTGTGGC-3’5'-CTGAGAAGGGCC A AGGTATTGTGGC-3 ' 1818
* 밑줄친 서열은 바꿔치기될 부분을 가리킨다. Underlined sequences indicate parts to be replaced.
전체 서열을 증폭하기 위한 프라이머로는 5’-GGTACCATGTCCACTGCGGTCCTGGAAAAC-3’(서열번호 19)과 5’-ATGCATTTACTTTATTTTCTGGAGGGCACTGCAAA-3’(서열번호 20)을 사용하였다. 주형으로 사용된 인간 PAH cDNA는 pMAL vector에 클로닝된 것으로 Prof. Aurora Martinez (University of Bergen, Norway)로부터 분양받은 것이다 (Martinez A, et al. 1995 Biochem J 306, 589597).5'- GGTACC ATGTCCACTGCGGTCCTGGAAAAC-3 '(SEQ ID NO: 19) and 5'- ATGCAT TTACTTTATTTTCTGGAGGGCACTGCAAA-3' (SEQ ID NO: 20) were used as primers to amplify the entire sequence. Human PAH cDNA used as a template was cloned into the pMAL vector. From Aurora Martinez (University of Bergen, Norway) (Martinez A, et al. 1995 Biochem J 306, 589597).
PCR 반응은 1X 반응 완충용액 (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 mM MgCl2, 0.2 mMd NTPs, 0.5 pmole 프라이머, 적당량의 주형 DNA, 2 unit의 pfu DNA 중합효소를 첨가하여 최종 50 μl로 맞추었다. 처음 95℃에서 5분간 DNA를 변성시킨 후 95℃에서 1분, 62℃에서 1분, 72℃에서 1분씩 30회 반복하여 증폭시켰다. 마지막으로 72℃에서 10분간 연장시간을 주어 PCR 반응을 종결시켰다. 0.7% 아가로스 겔에 전기영동하여 증폭된 DNA를 확인하였다.PCR reactions were performed using 1 × reaction buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 mM MgCl 2 , 0.2 mMd NTPs, 0.5 pmole primer, appropriate amount of template DNA, 2 units. pfu DNA polymerase was added to final 50 μl. The DNA was denatured for the first time at 95 ° C. for 5 minutes, and then amplified repeatedly for 1 minute at 95 ° C., 1 minute at 62 ° C. and 1 minute at 72 ° C. Finally, the PCR reaction was terminated by giving an extended time of 10 minutes at 72 ℃. The amplified DNA was confirmed by electrophoresis on a 0.7% agarose gel.
실시예 3. 발현벡터의 형질전환Example 3 Transformation of Expression Vectors
돌연변이 hPAH 유전자는 KpnI/NsiI 제한효소를 처리하여 분리하고 pDXA-3H 벡터(Manstein DJ, et al. 1995 Gene 162, 129134)에 삽입시켰다. 미리 배양한 세포성점균을 4℃에서 350xg로 3분간 원심분리하여 세포를 회수하였다. 회수된 세포는 차가운 전기천공(electroporation) 완충용액 (20 mM HEPES, 50 mM KCl, 10 mM NaCl, 1 mM MgSO4, 5mM NaHCO3, 1 mM NaH2PO4, pH7.0)으로 2회 씻어주어 배지를 제거한 후 전기천공 완충용액에 5×106 세포의 농도로 부유시켰다. 100 μl의 세포부유액에 10 μg의 형질전환용 DNA를 잘 섞어준 후 0.85 Kv로 2번 전기충격을 하고 얼음에 5분간 방치한 후 20 ml의 HL5가 들어있는 플레이트로 옮겨 22℃에서 배양하였다. 24시간 후 G418을 10 μg/ml의 농도로 첨가하였다. 배지와 항생제를 매일 교환하면서 광학현미경으로 형질전환 콜로니가 생성되는 것을 관찰하였다.Mutant hPAH gene was isolated by treatment with KpnI / NsiI restriction enzyme and inserted into pDXA-3H vector (Manstein DJ, et al. 1995 Gene 162, 129134). Cells were recovered by centrifuging the cell cultures previously cultured at 350 × g for 3 minutes at 4 ° C. The recovered cells were washed twice with cold electroporation buffer (20 mM HEPES, 50 mM KCl, 10 mM NaCl, 1 mM MgSO 4 , 5 mM NaHCO 3 , 1 mM NaH 2 PO 4 , pH7.0). After removing the medium, the cells were suspended in an electroporation buffer at a concentration of 5 × 10 6 cells. 10 μg of the DNA for transformation was well mixed with 100 μl of cell suspension, followed by two electric shocks at 0.85 Kv, and left on ice for 5 minutes, and then transferred to a plate containing 20 ml of HL5 and incubated at 22 ° C. After 24 hours G418 was added at a concentration of 10 μg / ml. Transforming colonies were observed by light microscopy with daily exchange of medium and antibiotics.
실시예 4. 생장속도 측정 및 PAH 활성 분석Example 4. Growth rate measurement and PAH activity assay
형질전환된 세포들을 우선 HL5 배지에서 2×106 세포/ml 수준까지 배양한 후 FM 배지로 씻어주고 다시 FM 배지에 1×106 세포/ml의 농도로 접종하였다. 22℃, 150 rpm의 진탕배양기(shaking incubator)에서 2일간 배양 후 세포계수기(hemacytometer)를 사용하여 세포수를 측정하고 8,000 rpm에서 5분간 원심분리하여 세포를 회수하였다.The transformed cells were first incubated in HL5 medium to 2 × 10 6 cells / ml, washed with FM medium, and then inoculated in FM medium at a concentration of 1 × 10 6 cells / ml. After culturing for 2 days in a shaking incubator at 22 ° C. and 150 rpm, the cell number was measured using a hemacytometer, and the cells were recovered by centrifugation at 8,000 rpm for 5 minutes.
회수한 세포는 100 μl의 용해 버퍼(50 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM PMSF)에 부유하였다. 액체질소를 이용하여 얼림과 녹임을 3회 반복하여 세포를 파쇄한 후 4℃, 15,000 rpm에서 20분간 원심 분리하여 상등액(조효소추출물)을 회수하였다. 회수한 조효소추출물을 세파덱스 G-25 스핀 칼럼을 사용하여 단백질을 회수하였다. 단백질의 농도 측정은 Bradford 방법을 이용하였고, 표준 단백질로는 BSA(bovine serum albumin)을 사용하였다.The recovered cells were suspended in 100 μl of lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM PMSF). After freezing and thawing three times using liquid nitrogen to crush the cells and centrifuged for 20 minutes at 4 ℃, 15,000 rpm to recover the supernatant (coenzyme extract). The recovered coenzyme extract was recovered protein using a Sephadex G-25 spin column. The concentration of protein was measured by Bradford method and BSA (bovine serum albumin) was used as a standard protein.
PAH 활성 분석은 50 μl의 반응액에 100 mM Tris-HCl (pH 7.5), 2 mM L-페닐알라닌, 100 유닛 카탈라아제, 5mM DTT, 0.4 mM BH4, 10 μg의 조효소추출물을 첨가하여 37℃에서 10분간 반응을 실시한 후 동량의 5%(v/v) 트리클로로아세트산(Trichloroacetic Acid) 용액으로 반응을 중단시켰다 (Kim HL, et al, 2012 FEBS Lett 586, 3596-3600). 13,000 rpm에서 10분간 원심분리하여 상등액에 존재하는 L-티로신의 양을 HPLC를 사용하여 정량분석하였다. HPLC에는 Gilson 321 Pump에 Rheodyne loop를 장착하여 사용하였으며, 역상의 Inertsil ODS-3 C18 (5 μm, 4.6x150 mm, GL sciences Ins.)을 사용하였다. 유동상으로는 30 mM 소듐 아세테이트(pH3.5)을 사용하여 1 ml/분의 속도로 흘려주었다. 티로신 피크(tyrosine peak)의 형광분석은 형광검출기(Fluorescence detector; RF-10A XL, Shimadzu)를 사용하여 290 nm/340 nm (excitation/emission) 파장과 Sens 1/Gain 2에서 검출하였다. PAH activity assay was performed by adding 100 mM Tris-HCl (pH 7.5), 2 mM L-phenylalanine, 100 unit catalase, 5 mM DTT, 0.4 mM BH4, 10 μg of coenzyme extract to 50 μl of the reaction solution for 10 minutes at 37 ° C. After the reaction, the reaction was stopped with the same amount of 5% (v / v) Trichloroacetic Acid solution (Kim HL, et al, 2012 FEBS Lett 586, 3596-3600). The amount of L-tyrosine present in the supernatant by centrifugation at 13,000 rpm for 10 minutes was quantified using HPLC. For HPLC, Rheodyne loop was attached to Gilson 321 Pump and reversed Inertsil ODS-3 C18 (5 μm, 4.6x150 mm, GL sciences Ins.) Was used. The fluid phase was flowed at a rate of 1 ml / min using 30 mM sodium acetate (pH 3.5). Fluorescence analysis of tyrosine peak was detected at 290 nm / 340 nm (excitation / emission) wavelength and Sens 1 / Gain 2 using a fluorescence detector (Fluorescence detector; RF-10A XL, Shimadzu).
실시예 5. 웨스턴 블롯 분석Example 5. Western Blot Analysis
조효소추출물(50 μg)을 5X 샘플 버퍼 (250 mM Tris-HCl pH 6.8, 10% SDS, 30% glycerol, 5% β-mercapitalethanol, 0.02% bromophenol blue)와 섞은 후 SDS-PAGE와 웨스턴 블로팅을 실시하였다. SDS-PAGE 후, 트랜스퍼 버퍼 (25 mM Tris, 192 mM glycine, pH 8.3, 10% Methanol)를 사용하여 45 volt에서 2시간 전기영동하여 단백질을 니트로셀룰로오스 멤브레인에 이동시켰다. 트랜스퍼가 끝난 멤브레인을 10 ml의 TTBS (1X TBS-10 mM Tris, 150 mM NaCl, pH 7.5에 500 μl의 Tween-20을 첨가) 용액으로 씻어주고, 10 ml의 블로킹 (10 ml의 TTBS 용액에 5% BSA를 첨가) 용액으로 1시간 동안 가볍게 흔들면서 반응시켰다. 10 ml의 TTBS 용액으로 씻어준 후 10 ml의 TTBS 용액에 5 μl의 1차 항체(인간 PAH 항체; Abcam)를 첨가하여 가볍게 흔들면서 하룻밤 방치하였다가 10 ml의 TTBS 용액으로 5분간 두 번 씻어주었다. 다시 10 ml의 TTBS 용액에 2.5 μl의 2차 항체(horseradish peroxidase-conjugated secondary antibodies)를 첨가하여 1시간 동안 반응시킨 후 10 ml의 TTBS 용액으로 5분간 세 번 씻어주었다. 멤브레인을 OHP 필름 위에 놓고 2 ml의 ECL 용액을 멤브레인에 올려 3분간 반응 후 용액을 휴지에 흡수시켜 제거하였다. 멤브레인 위에 다시 OHP 필름을 겹친 후 멤브레인과 필름 사이의 기포를 제거하고 이미징 장비인 Fusion-SL4 Spectra (Vilber, Germany)를 이용해 밴드를 검출하였다.Coenzyme extract (50 μg) was mixed with 5X sample buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 30% glycerol, 5% β-mercapitalethanol, 0.02% bromophenol blue), followed by SDS-PAGE and Western blotting. It was. After SDS-PAGE, proteins were transferred to nitrocellulose membranes by electrophoresis at 45 volt for 2 hours using transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 10% Methanol). The transferred membrane was washed with 10 ml of TTBS (1X TBS-10 mM Tris, 150 mM NaCl, pH 7.5 added 500 μl Tween-20) solution, and 10 ml of blocking (5 in 10 ml TTBS solution). The reaction was stirred gently for 1 hour with a solution). After washing with 10 ml of TTBS solution, 5 μl of primary antibody (human PAH antibody; Abcam) was added to 10 ml of TTBS solution, left overnight with gentle shaking, and then washed twice with 10 ml of TTBS solution for 5 minutes. . Then, 2.5 μl secondary antibody (horseradish peroxidase-conjugated secondary antibodies) was added to the 10 ml TTBS solution for 1 hour and then washed three times for 5 minutes with 10 ml TTBS solution. The membrane was placed on an OHP film and 2 ml of ECL solution was placed on the membrane to react for 3 minutes, and then the solution was absorbed into a tissue and removed. After overlapping the OHP film on the membrane again, the bubble between the membrane and the film was removed, and the band was detected using Fusion-SL4 Spectra (Vilber, Germany).
실험결과 1. 생장속도와 PAH 활성 간의 상관관계 Experimental Results 1. Correlation between Growth Rate and PAH Activity
PAH 유전자가 치환된 돌연변이 세포성점균(pah -)에 야생형을 비롯하여 돌연변이 인간 PAH 유전자 각각이 삽입된 발현벡터를 넣은 형질전환체 9가지를 만들었다. 이 형질전환체들을 FM 배지에서 48시간 키운 후 생장속도를 비교하였다 (도 1의 A). PAH gene is substituted with mutant cells Slime Molds (pah -) as well as wild-type to the created transformants 9 kinds of each mutant human PAH gene inserted into the expression vector. These transformants were grown in FM medium for 48 hours and then growth rate was compared (A in FIG. 1).
도 1의 A에 나타난 바와 같이, pah - 균주는 FM 배지에서 전혀 생장할 수 없는 것과 달리, 야생형 균주(WT) 뿐만 아니라 돌연변이 형질전환체들은 정도의 차이는 있지만 모두 생장하였다. 특히 심각한 PKU를 야기시키는 돌연변이로 알려진 S349L의 형질전환체는 가장 낮은 생장을 보여주었으며, PAH 활성도 간신히 측정되는 수준이었다. 전체적으로 형질전환체 마다 생장속도와 PAH 활성의 수준은 상관관계를 나타내었으며, 그 수준은 PKU 증상의 심각성과 관련이 있음을 시사하였다. 이는 생장속도와 PAH 활성 간에 밀접한 상관관계가 있음을 보여주는 것으로 이를 확인하기 위해 선형회귀분석을 실시하였다. As shown in A of FIG. 1, the pah − strain was not able to grow at all in the FM medium, but the wild type strain (WT) as well as the mutant transformants were all grown to some extent. In particular, S349L transformants, known as mutations that cause severe PKU, showed the lowest growth, and PAH activity was barely measured. Overall, growth rate and level of PAH activity were correlated with each transformant, suggesting that the level was related to the severity of PKU symptoms. This shows a close correlation between growth rate and PAH activity. A linear regression analysis was performed to confirm this.
도 1의 B에서, 선형상관관계는 통계적으로 유의성이 있음을 보여줌으로써 생장속도가 PAH 활성에 의존함을 지지하였다. 이러한 결과는 pah -에서 상보적 발현을 통해 그 생장속도로서 미스센스 돌연변이를 가진 인간 PAH를 정량적으로 평가할 수 있음을 제시한다. 생장속도는 PAH 활성보다 측정하기가 용이하다는 장점이 있다.In FIG. 1B, linear correlation was shown to be statistically significant, supporting the growth rate dependent on PAH activity. These results suggest that complementary expression at pah − can quantitatively assess human PAH with missense mutations as its growth rate. Growth rate has the advantage of being easier to measure than PAH activity.
실험결과 2. 세포내 테트라히드로테린 농도가 단백질 잔존량에 미치는 영향 Experimental Results 2. Effect of Intracellular Tetrahydroterin Concentration on Protein Remaining Amount
pah -에서 발현된 돌연변이 PAH 단백질량을 측정하기 위하여 조효소추출물을 가지고 웨스턴 블롯 분석을 실시하였다. pah - to provide a measure of the amount of mutant PAH protein expressed in a with a crude enzyme extract was subjected to Western blot analysis.
도 2의 A I에서와 같이, 예상대로 돌연변이에 따라 단백질량에 차이를 보였다. K42I, L48S, S349L, R408W의 경우는 야생형의 10% 미만이었으며, 다른 돌연변이들은 50% 이상으로 나타났다. 돌연변이에 따라 단백질량에서의 차이가 있다는 것은 세포성점균에서 사람 PAH가 외부유래 단백질로서 무작위적인 분해를 당하는 것이 아니라, 인간세포에서와 유사한 단백질 접힘에서의 문제와 대면하고 있음을 의미한다.As shown in A I of FIG. 2, the protein amount was varied according to the mutation as expected. K42I, L48S, S349L, and R408W were less than 10% of wild type, and other mutations were more than 50%. The difference in protein amount by mutation means that human PAH is not subject to random degradation as an exogenous protein in cellular bacteria, but faces a similar problem in protein folding as in human cells.
pah -는 야생형과 동일하게 많은 양의 테트라히드로테린을 생성하기 때문에 돌연변이 PAH 단백질들은 이들에 의한 pharmacological chaperone 효과를 받고 있다고 추정된다. 이러한 효과를 검증하기 위하여 돌연변이 PAH 단백질들을 세피아테린 환원효소가 결핍된 돌연변이 균주(spr -)에 발현시켰다. 이 균주에는 세포성점균의 PAH가 그대로 남아있기 때문에 인간 PAH의 단백질량만을 분석하였다. Since pah − produces the same amount of tetrahydroterin as the wild type, mutant PAH proteins are presumed to have the pharmacological chaperone effect by them. To verify this effect, mutant PAH proteins were expressed in mutant strains ( spr − ) deficient in sepiaterin reductase. Since the PAH of the cellular mycobacteria remained in this strain, only the protein amount of human PAH was analyzed.
도 2의 A II에서와 같이, F39L, K42I, I65T의 경우는 단백질량이 현저하게 감소하였다. 흥미롭게도 이 돌연변이들은 BH4-반응성이 있다고 알려진 것들이다 (Zurfluh MR, et al. 2008 Hum Mutat 29, 167-175). 심각하지는 않지만, R252Q와 L255V도 절반 수준으로 단백질량이 감소하였다. 예상 밖으로 S349L은 비교적 높게 단백질량이 증가하였다. 이러한 결과는 돌연변이 인간 PAH의 BH4에 대한 반응성을 세포성점균을 이용하여 세포 수준에서 연구할 수 있음을 의미한다.As in A II of FIG. 2, in the case of F39L, K42I, and I65T, the amount of protein was significantly reduced. Interestingly, these mutations are known to be BH4-reactive (Zurfluh MR, et al. 2008 Hum Mutat 29, 167-175). Although not severe, R252Q and L255V also decreased in half. Unexpectedly, S349L increased the amount of protein relatively high. These results indicate that the reactivity of mutant human PAH to BH4 can be studied at the cellular level using cellular slime molds.
실시결과 3. 영양배지가 단백질 잔존량에 미치는 영향 Result 3. Effect of nutritional medium on protein residual
상기 결과는 테트라히드로테린과 유사한 효과를 발휘하는 화합물을 세포성점균을 이용하여 스크리닝할 수 있음을 제시하였다. 그 가능성을 타진하기 위하여 pah -를 영양배지인 HL5 배지에서 배양하고 단백질량을 분석하였다 (도 2의 A III). HL5 배지에 포함되는 효모추출물에는 천연성분이 풍부하게 존재하고 있다. The results suggest that compounds exhibiting similar effects to tetrahydroterin can be screened using cellular slime molds. To assess the possibility, pah − was cultured in HL5 medium, a nutrient medium, and the amount of protein was analyzed (A III in FIG. 2). Yeast extracts contained in the HL5 medium are rich in natural ingredients.
도 2의 C에서, 단백질량의 증가를 보여주는 돌연변이는 L48S, S349L, R408W였으며, 이들 중에서 S349L은 야생형 수준으로 회복되었다. 또한 단백질량의 증가가 효소활성과 연결되는지 알아본 결과, 도 2의 D와 같이, S349L과 R408W에서 유의한 활성 증가가 관찰되었다. S349L의 경우 단백질량에 비례하는 활성증가를 보여주지 않았지만 FM 배지에서와 비교하여 5배 수준으로 증가하였다. 이는 아마도 S349L 돌연변이가 단백질 촉매활성에 심각한 영향을 미치기 때문으로 사료되며, 이전의 연구결과 (Gamez A, et al. 2000 J Biol Chem 275, 29737-29742)도 이를 지지하고 있다. R408W의 경우는 단백질량과 효소활성 모두 유사한 (2배) 수준으로 증가하였다. 효모추출물 내에 어떤 성분이 S349L과 R408W의 단백질 안정성에 영향을 미쳤는지는 확인되지 않았으나, 이 결과는 세포성점균을 이용하여 일부 돌연변이에 특이적인 천연화합물 또는 약물 샤프론을 찾아내는데 이용할 수 있음을 보여준다.In FIG. 2C, mutations showing an increase in protein amount were L48S, S349L, R408W, of which S349L recovered to wild-type levels. In addition, as a result of examining whether the increase in protein amount is linked to the enzyme activity, as shown in D of Figure 2, significant increase in activity was observed in S349L and R408W. S349L did not show an increase in activity proportional to the amount of protein but increased to a 5-fold level compared to that in FM medium. This is probably because S349L mutations have a significant effect on protein catalytic activity, and previous studies (Gamez A, et al. 2000 J Biol Chem 275, 29737-29742). In the case of R408W, both protein and enzyme activity increased to similar (twice) levels. It has not been determined which components in yeast extracts have influenced the protein stability of S349L and R408W, but the results show that the cellular bacilli can be used to find natural compounds or drug saffrons specific for some mutations.
추가적으로, HL5 배지에서 배양한 S349L 균주에서 PAH 단백질량이 회복되는 것이 HL5 배지에 포함된 효모추출물에 의한 것인지 확인하기 위하여, S349L 균주를 효모추출물이 첨가된 FM 배지에서 키운 후 웨스턴 블롯 분석을 수행하였다. 도 4는 웨스턴 블롯 결과로서, 상단의 사진은 웨스턴 블롯의 결과이고 하단의 그래프는 정량값을 나타낸다. 효모추출물 1X의 농도는 리터당 5 g이었다. 도 4에 나타난 결과에 따르면, 효모추출물에 S349L에 약리학적 샤프론과 같이 단백질 안정성을 증가시키는 성분이 존재함이 확인되었다. In addition, in order to confirm whether the recovery of PAH protein amount from the S349L strain cultured in HL5 medium was caused by the yeast extract included in the HL5 medium, the S349L strain was grown in the FM medium to which the yeast extract was added, and then Western blot analysis was performed. 4 is a Western blot result, the photo at the top shows the result of the Western blot and the graph at the bottom shows the quantitative value. The concentration of yeast extract 1 × was 5 g per liter. According to the results shown in Figure 4, it was confirmed that there is a component in the yeast extract that increases protein stability, such as pharmacological saffron in S349L.
결과 분석Result analysis
PKU와 관련된 돌연변이 PAH를 세포성점균에서 발현시킨 것은 본 발명이 처음이다. 따라서 세포성점균을 이용한 발현시스템이 인간 PAH 연구에 적합한지를 검증하기 위하여, 본 발명을 통해 얻어진 결과를 인간세포를 비롯한 다른 발현시스템에서 연구된 결과와 비교하였다. 연구 결과를 설명하는데 편의를 도모하기 위해 효소 활성과 단백질량 간의 관계를 보여주는 그래프를 준비하였다 (도 3). It is the first time that the mutant PAH associated with PKU is expressed in cellular slime molds. Therefore, in order to verify whether the expression system using the cellular slime mold is suitable for the study of human PAH, the results obtained through the present invention were compared with those studied in other expression systems including human cells. For convenience in explaining the results of the study, a graph showing the relationship between the enzyme activity and the amount of protein was prepared (FIG. 3).
도 3은 돌연변이가 단백질 안정성과 촉매활성에 미치는 영향을 보여준다. 궁극적으로 개개의 돌연변이 인간 PAH의 표현형을 결정하는데 관여하는 세포내 PAH 활성은 단백질량과 그 촉매활성에 의해 결정된다. 도 3에서 원점으로부터 WT을 지나는 선을 그렸다. 이것은 야생형 인간 PAH의 진정한 특이활성을 보여주는 것으로 WT line이라 명명하였다. 오로지 단백질 안정성에만 영향을 미치는 돌연변이를 가정한다면 이것은 WT line을 따라서 움직일 것이다. 만약 돌연변이가 단백질 안정성과 촉매활성 둘 다에 영향을 준다면 이 돌연변이는 WT line 아래에 나타날 것이다. 두 가지 각각에 미치는 돌연변이의 영향력에 따라 위치가 변할 것이며, 심각한 돌연변이일수록 원점에 가까워질 것이다. 3 shows the effect of mutations on protein stability and catalytic activity. Ultimately, the intracellular PAH activity involved in determining the phenotype of individual mutant human PAHs is determined by the amount of protein and its catalytic activity. In FIG. 3, a line passing from the origin through the WT is drawn. This shows the true specific activity of wild-type human PAH and named it WT line. Assuming a mutation that only affects protein stability, it will move along the WT line. If the mutation affects both protein stability and catalytic activity, the mutation will appear below the WT line. The position will change depending on the influence of the mutation on each of the two, and the more severe the mutation, the closer to the origin.
도 3에서, R252Q, S349L, R408W는 WT line 아래에 위치하고 있으며 이는 이전에 보고된 연구결과에 부합한다. K42I는 WT line에 붙어있지만 조절 도메인 돌연변이에 속하는 다른 것들과 유사하기 때문에 아래에서 함께 논의되었다. 도 3에서, R252는 전형적으로 촉매활성에 문제를 야기시키는 돌연변이로 보이며 다른 발현시스템에서 연구된 결과도 이를 지지한다. In Figure 3, R252Q, S349L, R408W are located below the WT line, which is consistent with the previously reported study results. K42I is discussed below because it is attached to the WT line but similar to others belonging to regulatory domain mutations. In FIG. 3, R252 typically appears to be a mutation that causes problems with catalytic activity and the results studied in other expression systems support this.
R252는 촉매 도메인에 위치하고 있으며 이 아미노산이 바뀌면 도메인에서 안정적인 상호작용을 파괴시킬 것으로 추정되고 있다 (Erlandsen H, et al. 2003 Pediatrics 112, 1557-1565). 대장균과 in vitro 발현시스템에서 얻어진 R252Q 재조합 단백질은 야생형의 3~11.4% 활성을 나타내었다 (Bjørgo E, et al. 1998 Eur J Biochem 257, 1-10). R252 is located in the catalytic domain and it is believed that alteration of this amino acid will disrupt stable interactions in the domain (Erlandsen H, et al. 2003 Pediatrics 112, 1557-1565). R252Q recombinant protein obtained from E. coli and in vitro expression system showed 3 ~ 11.4% activity of wild type (Bjørgo E, et al. 1998 Eur J Biochem 257, 1-10).
S349L은 단백질 안정성과 촉매활성 둘 다를 심각하게 손상시킨다고 알려져 있으며 (Gamez A, et al. 2000 J Biol Chem 275, 29737-29742), 도 2의 C와 D에서 보여준 결과와도 일치한다. S349L is known to seriously impair both protein stability and catalytic activity (Gamez A, et al. 2000 J Biol Chem 275, 29737-29742), which is consistent with the results shown in FIGS.
COS 세포에서 발현된 R408W에 대한 연구결과는 이 돌연변이가 주로 단백질안정성에 문제가 있음을 보여주었다 (Pey AL, et al. 2003 Hum Mutat 21, 370-378). 도 3에서, 단백질량에 비해 낮은 활성을 가지는 것으로 나타났으나, FM과 HL5 배지에서의 결과를 비교한 도 2의 C와 D에서는 단백질량의 증가와 더불어 효소 활성도 비례하여 증가함을 보여준다.Studies on R408W expressed in COS cells have shown that this mutation is primarily problematic for protein stability (Pey AL, et al. 2003 Hum Mutat 21, 370-378). In Figure 3, it was shown to have a lower activity compared to the protein amount, but in the C and D of Figure 2 comparing the results in FM and HL5 medium shows that the enzyme activity is also proportionally increased with the increase in the protein amount.
반면에 F39L, L48S, I65T, L255V는 WT line 위에서 발견되고 있다. K42I는 WT line 상에 위치하지만 같은 특성을 가지는 돌연변이에 해당된다. 이 그룹은 야생형보다 단백질 안정성은 낮지만 촉매활성은 높다는 것을 의미한다. 이런 경우는 일반적이지 않지만 L255V를 제외하고 나머지는 조절 도메인에 위치하고 있다. 불활성 상태의 인간 PAH에서 조절 도메인은 활성자리를 덮고 있는 상태로 억제기능을 수행한다고 알려져 있다 (Fitzpatrick PF, 2012 Arch Biochem Biophys 519, 194201). 따라서 조절 도메인에서의 돌연변이는 촉매활성을 오히려 증가시키는 효과를 발휘할 수 있다. F39L, L48S, I65T and L255V, on the other hand, are found on the WT line. K42I is a mutation located on the WT line but with the same characteristics. This group means lower protein stability than wild type but higher catalytic activity. This is not common, but the rest is in the regulatory domain except for L255V. In inactive human PAHs, regulatory domains are known to perform inhibitory functions by covering active sites (Fitzpatrick PF, 2012 Arch Biochem Biophys 519, 194201). Thus, mutations in the regulatory domain may exert an effect of increasing catalytic activity.
in vitro 발현 결과에 따르면 (Waters PJ, et al. 2000 Mol Genet Metab 69, 101-110), 대장균에서 발현된 F39L, K42I, L48S, I65T 효소들은 각각 야생형의 114%, 133%, 84%, 92% 활성을 보여주었다. 더군다나 TNT-T7 rabbit reticulocyte system에서 이들의 단백질 분해속도는 L48S, K42I, F39L, I65T, WT 순으로 나타났으며, 이 결과는 인간 신장세포에서도 확인되었다. 이러한 결과는 단백질량들을 분석한 결과 (도 2의 A와 B)와도 일치한다. 다만 L255V는 이전의 다른 발현시스템에서 연구된 결과 (Bjørgo E, et al. 1998 Eur J Biochem 257, 1-10) 보다 높은 단백질 안정성과 촉매활성을 보여주었는데, 이러한 차이는 세포성점균의 배양이 22℃에서 이루어지기 때문인 것으로 보인다. 따라서 전체적으로 본 발명의 결과가 다른 발현시스템에서 얻어진 결과에 잘 부합됨이 확인되었다.In vitro expression results (Waters PJ, et al. 2000 Mol Genet Metab 69, 101-110), E. coli F39L, K42I, L48S, I65T enzymes were 114%, 133%, 84%, 92 of wild type, respectively % Activity was shown. In addition, the protein degradation rate in the TNT-T7 rabbit reticulocyte system was in the order of L48S, K42I, F39L, I65T, WT, and the results were confirmed in human kidney cells. This result is also consistent with the results of analyzing protein amounts (A and B of FIG. 2). However, L255V showed higher protein stability and catalytic activity than previously studied in other expression systems (Bjørgo E, et al. 1998 Eur J Biochem 257, 1-10). It seems to be because it is made at ℃. Therefore, it was confirmed that the results of the present invention were in good agreement with those obtained in other expression systems as a whole.
결론적으로, 본 발명에 따르면 다른 발현시스템을 대체하여 세포성점균이 인간 PAH에서 미스센스 돌연변이를 단백질과 세포 수준에서 분석할 수 있는 유용한 발현시스템임을 보여주었다. 따라서 인간 PAH의 미스센스 돌연변이는 세포성점균에서의 발현을 통해 그 생장속도로서 정량적으로 평가될 수 있으며, 좀더 in vivo에 가까운 조건에서 단백질 안정성과 촉매 활성을 돌연변이 단백질들의 특성을 연구할 수 있게 해준다. 무엇보다도 세포수준의 분석(cell-based assay)이 가능함으로써 구조적으로 불안정한 돌연변이 단백질에 대한 연구를 가능하게 해준다. 세포성점균은 경제적인 연구모델인 동시에 안정적으로 유지되는 발현 시스템으로서 돌연변이 단백질들에 대한 상세한 연구를 가능케할 것으로 생각되며, 더군다나 테트라히드로테린과 다른 후보분자들의 pharmacological chaperone 효과를 분석할 수 있는 기회를 제공할 것이다. 또는 페닐케톤뇨증 환자에게 영향을 줄 수 있는 약물이나 음식물 성분의 분석에도 이용될 것으로 기대된다. 이를 통해 돌연변이 유형에 특이적인 환자맞춤형 치료에 접근할 수 있는 유용한 연구시스템으로서도 중요한 의미를 가진다. 더나아가서는 단백질 접힘과 관련된 많은 질환에도 응용이 가능할 것으로 기대된다.In conclusion, in accordance with the present invention, it has been shown that, in place of other expression systems, the cellular slime mold is a useful expression system capable of analyzing missense mutations at the protein and cellular levels in human PAH. Thus, missense mutations in human PAH can be assessed quantitatively as their growth rate through expression in cellular slime molds, allowing for the characterization of mutant proteins for protein stability and catalytic activity under more in vivo conditions. . First of all, cell-based assays enable the study of structurally unstable mutant proteins. Cellular slime molds are an economical research model and a stable expression system that will enable the detailed study of mutant proteins and furthermore provide an opportunity to analyze the pharmacological chaperone effects of tetrahydroterin and other candidate molecules. Will provide. It is also expected to be used for analysis of drugs and food components that may affect patients with phenylketonuria. This is also important as a useful research system that provides access to patient-specific therapies specific to mutation types. Furthermore, it is expected to be applicable to many diseases related to protein folding.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.
1. 돌연변이 PAH 연구시스템으로서의 세포성점균의 적합성 검증: 8가지 돌연변이 단백질들의 활성과 단백질량 분석을 통해 인간세포를 비롯한 다양한 발현시스템에서 연구된 결과와 유사한 결과 도출.1. Verification of the cellular bacillus as a mutant PAH research system: Activity and protein content analysis of 8 mutant proteins yielded results similar to those studied in various expression systems including human cells.
2. 세포수준의 대량분석 시스템: 생장속도를 이용한 정량적 분석이 가능하며, 특히 구조적으로 불안정한 돌연변이 PAH에 대한 연구 가능.2. Cell-level mass spectrometry systems: quantitative analysis using growth rates, especially for structurally unstable mutant PAH.
3. 단백질 수준의 분석 시스템: 정제과정 없이도 돌연변이 PAH에 대한 연구 가능. 3. Protein level analysis system: mutant PAHs can be studied without purification.
4. 경제적이고 안정적인 발현시스템: 개개의 PKU 돌연변이 단백질에 대한 심화 연구 가능. 세포성점균은 대장균과 유사한 배지를 사용하므로 포유동물세포 배양에 비해 저렴.4. Economical and stable expression system: Further research on individual PKU mutant proteins is possible. Cellular slime is cheaper than mammalian cell culture because it uses a medium similar to E. coli.
5. 환자 맞춤 치료에 이용: PKU 환자의 돌연변이 유전형에 집중된 연구 가능.5. Use in patient-specific treatment: focused research on mutant genotypes in PKU patients.
<110> inje university industry-academic cooperation foundation<110> inje university industry-academic cooperation foundation
<120> Analysis method of activity for human phenylalanine hydroxylase using Dictyostelium discoideum<120> Analysis method of activity for human phenylalanine hydroxylase using Dictyostelium discoideum
<130> NP14-1354<130> NP14-1354
<160> 20<160> 20
<170> KopatentIn 2.0<170> KopatentIn 2.0
<210> 1<210> 1
<211> 452<211> 452
<212> PRT<212> PRT
<213> homo sapiens<213> homo sapiens
<400> 1<400> 1
Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu SerMet Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu Ser
1 5 10 15   1 5 10 15
Asp Phe Gly Gln Glu Thr Ser Tyr Ile Glu Asp Asn Cys Asn Gln AsnAsp Phe Gly Gln Glu Thr Ser Tyr Ile Glu Asp Asn Cys Asn Gln Asn
20 25 30              20 25 30
Gly Ala Ile Ser Leu Ile Phe Ser Leu Lys Glu Glu Val Gly Ala LeuGly Ala Ile Ser Leu Ile Phe Ser Leu Lys Glu Glu Val Gly Ala Leu
35 40 45          35 40 45
Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val Asn Leu Thr HisAla Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val Asn Leu Thr His
50 55 60      50 55 60
Ile Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp Glu Tyr Glu Phe PheIle Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp Glu Tyr Glu Phe Phe
65 70 75 80  65 70 75 80
Thr His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Ile LysThr His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Iles Lys
85 90 95                  85 90 95
Ile Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg AspIle Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg Asp
100 105 110             100 105 110
Lys Lys Lys Asp Thr Val Pro Trp Phe Pro Arg Thr Ile Gln Glu LeuLys Lys Lys Asp Thr Val Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu
115 120 125         115 120 125
Asp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp AlaAsp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala
130 135 140     130 135 140
Asp His Pro Gly Phe Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys GlnAsp His Pro Gly Phe Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln
145 150 155 160 145 150 155 160
Phe Ala Asp Ile Ala Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro ArgPhe Ala Asp Ile Ala Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg
165 170 175                 165 170 175
Val Glu Tyr Met Glu Glu Glu Lys Lys Thr Trp Gly Thr Val Phe LysVal Glu Tyr Met Glu Glu Glu Lys Lys Thr Trp Gly Thr Val Phe Lys
180 185 190             180 185 190
Thr Leu Lys Ser Leu Tyr Lys Thr His Ala Cys Tyr Glu Tyr Asn HisThr Leu Lys Ser Leu Tyr Lys Thr His Ala Cys Tyr Glu Tyr Asn His
195 200 205         195 200 205
Ile Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp Asn IleIle Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp Asn Ile
210 215 220     210 215 220
Pro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr Gly PhePro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr Gly Phe
225 230 235 240 225 230 235 240
Arg Leu Arg Pro Val Ala Gly Leu Leu Ser Ser Arg Asp Phe Leu GlyArg Leu Arg Pro Val Ala Gly Leu Leu Ser Ser Arg Asp Phe Leu Gly
245 250 255                 245 250 255
Gly Leu Ala Phe Arg Val Phe His Cys Thr Gln Tyr Ile Arg His GlyGly Leu Ala Phe Arg Val Phe His Cys Thr Gln Tyr Ile Arg His Gly
260 265 270             260 265 270
Ser Lys Pro Met Tyr Thr Pro Gln Pro Asp Ile Cys His Glu Leu LeuSer Lys Pro Met Tyr Thr Pro Gln Pro Asp Ile Cys His Glu Leu Leu
275 280 285         275 280 285
Gly His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser GlnGly His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln
290 295 300     290 295 300
Glu Ile Gly Leu Ala Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu LysGlu Ile Gly Leu Ala Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu Lys
305 310 315 320 305 310 315 320
Leu Ala Thr Ile Tyr Trp Phe Thr Val Glu Phe Gly Leu Cys Lys GlnLeu Ala Thr Ile Tyr Trp Phe Thr Val Glu Phe Gly Leu Cys Lys Gln
325 330 335                 325 330 335
Gly Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe GlyGly Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly
340 345 350             340 345 350
Glu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lys Leu Leu Pro Leu GluGlu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lys Leu Leu Pro Leu Glu
355 360 365         355 360 365
Leu Glu Lys Thr Ala Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln ProLeu Glu Lys Thr Ala Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro
370 375 380     370 375 380
Leu Tyr Tyr Val Ala Glu Ser Phe Asn Asp Ala Lys Glu Lys Val ArgLeu Tyr Tyr Val Ala Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg
385 390 395 400 385 390 395 400
Asn Phe Ala Ala Thr Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp ProAsn Phe Ala Ala Thr Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro
405 410 415                 405 410 415
Tyr Thr Gln Arg Ile Glu Val Leu Asp Asn Thr Gln Gln Leu Lys IleTyr Thr Gln Arg Ile Glu Val Leu Asp Asn Thr Gln Gln Leu Lys Ile
420 425 430             420 425 430
Leu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala LeuLeu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala Leu
435 440 445         435 440 445
Gln Lys Ile LysGln Lys Ile Lys
450     450
<210> 2<210> 2
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atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60
gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120
ctcaaagaag aagttggtgc attggccaaa gtattgcgct tatttgagga gaatgatgta 180ctcaaagaag aagttggtgc attggccaaa gtattgcgct tatttgagga gaatgatgta 180
aacctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240aacctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240
acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300
gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360
ttcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420ttcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420
gaactggatg ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480gaactggatg ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480
tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540
gaggaagaaa agaaaacatg gggcacagtg ttcaagactc tgaagtcctt gtataaaacc 600gaggaagaaa agaaaacatg gggcacagtg ttcaagactc tgaagtcctt gtataaaacc 600
catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660
gaagataaca ttccccagct ggaagacgtt tctcaattcc tgcagacttg cactggtttc 720gaagataaca ttccccagct ggaagacgtt tctcaattcc tgcagacttg cactggtttc 720
cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780
cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840
cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900
cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag 960cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag 960
ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020
aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080
aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140
gagttccagc ccctgtatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200gagttccagc ccctgtatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200
aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260aactttgctg ccacaatacc tcggccctct tcagttcgct acgacccata cacccaaagg 1260
attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320
attggaatcc tttgcagtgc cctccagaaa ataaagtaa 1359attggaatcc tttgcagtgc cctccagaaa ataaagtaa 1359
<210> 3<210> 3
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for F39L<223> forward PCR primer for F39L
<400> 3<400> 3
ccatatcact gatcttgtca ctcaaagaag aa 32ccatatcact gatcttgtca ctcaaagaag aa 32
<210> 4<210> 4
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for F39L<223> reverse PCR primer for F39L
<400> 4<400> 4
ttcttctttg agtgacaaga tcagtgatat gg 32ttcttctttg agtgacaaga tcagtgatat gg 32
<210> 5<210> 5
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for K42I<223> forward PCR primer for K42I
<400> 5<400> 5
ctgatcttct cactcataga agaagttggt gc 32ctgatcttct cactcataga agaagttggt gc 32
<210> 6<210> 6
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for K42I<223> reverse PCR primer for K42I
<400> 6<400> 6
gcaccaactt cttctatgag tgagaagatc ag 32gcaccaactt cttctatgag tgagaagatc ag 32
<210> 7<210> 7
<211> 26<211> 26
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for L48S<223> forward PCR primer for L48S
<400> 7<400> 7
gaagttggtg catcggccaa agtatt 26gaagttggtg catcggccaa agtatt 26
<210> 8<210> 8
<211> 26<211> 26
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for L48S<223> reverse PCR primer for L48S
<400> 8<400> 8
aatactttgg ccgatgcacc aacttc 26aatactttgg ccgatgcacc aacttc 26
<210> 9<210> 9
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for I65T<223> forward PCR primer for I65T
<400> 9<400> 9
gtaaacctga cccacactga atctagacct tc 32gtaaacctga cccacactga atctagacct tc 32
<210> 10<210> 10
<211> 32<211> 32
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for I65T<223> reverse PCR primer for I65T
<400> 10<400> 10
gaaggtctag attcagtgtg ggtcaggttt ac 32gaaggtctag attcagtgtg ggtcaggttt ac 32
<210> 11<210> 11
<211> 27<211> 27
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for R252Q<223> forward PCR primer for R252Q
<400> 11<400> 11
tgctttcctc tcaggatttc ttgggtg 27tgctttcctc tcaggatttc ttgggtg 27
<210> 12<210> 12
<211> 27<211> 27
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for R252Q<223> reverse PCR primer for R252Q
<400> 12<400> 12
cacccaagaa atcctgagag gaaagca 27cacccaagaa atcctgagag gaaagca 27
<210> 13<210> 13
<211> 23<211> 23
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for L255V<223> forward PCR primer for L255V
<400> 13<400> 13
ctcgggattt cgtgggtggc ctg 23ctcgggattt cgtgggtggc ctg 23
<210> 14<210> 14
<211> 23<211> 23
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for L255V<223> reverse PCR primer for L255V
<400> 14<400> 14
caggccaccc acgaaatccc gag 23caggccaccc acgaaatccc gag 23
<210> 15<210> 15
<211> 28<211> 28
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for S349L<223> forward PCR primer for S349L
<400> 15<400> 15
ctgggctcct gttatccttt ggtgaatt 28ctgggctcct gttatccttt ggtgaatt 28
<210> 16<210> 16
<211> 28<211> 28
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for S349L<223> reverse PCR primer for S349L
<400> 16<400> 16
aattcaccaa aggataacag gagcccag 28aattcaccaa aggataacag gagcccag 28
<210> 17<210> 17
<211> 25<211> 25
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for R408W<223> forward PCR primer for R408W
<400> 17<400> 17
gccacaatac cttggccctt ctcag 25gccacaatac cttggccctt ctcag 25
<210> 18<210> 18
<211> 25<211> 25
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for R408W<223> reverse PCR primer for R408W
<400> 18<400> 18
ctgagaaggg ccaaggtatt gtggc 25ctgagaaggg ccaaggtatt gtggc 25
<210> 19<210> 19
<211> 30<211> 30
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> forward PCR primer for hPAH cDNA<223> forward PCR primer for hPAH cDNA
<400> 19<400> 19
ggtaccatgt ccactgcggt cctggaaaac 30ggtaccatgt ccactgcggt cctggaaaac 30
<210> 20<210> 20
<211> 35<211> 35
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> reverse PCR primer for hPAH cDNA<223> reverse PCR primer for hPAH cDNA
<400> 20<400> 20
atgcatttac tttattttct ggagggcact gcaaa 35atgcatttac tttattttct ggagggcact gcaaa 35

Claims (11)

  1. a) 서열번호 1의 아미노산 서열로 표시되는 인간 PAH(phenylalanine hydroxylase)를 코딩하는 유전자에서 페닐케톤뇨증(phenylketonuria)을 야기시키는 돌연변이를 포함하는, 돌연변이 PAH 유전자를 세포성점균에 형질전환 시키는 단계;a) transforming a mutant PAH gene into a cellular bacterium, the mutation comprising a mutation causing phenylketonuria in a gene encoding human phenylalanine hydroxylase (PAH) represented by the amino acid sequence of SEQ ID NO: 1;
    b) 상기 돌연변이 PAH 유전자로 치환된 세포성점균을 티로신(tyrosine)이 결핍된 최소영양배지에서 배양시키는 단계; 및b) culturing the cellular slime mold substituted with the mutated PAH gene in a minimal nutrient medium lacking tyrosine; And
    c) 상기 배양되는 세포성점균의 생장 속도를 측정하는 단계를 포함하는 세포성점균을 이용한 인간 돌연변이 PAH 활성분석 방법.c) Human mutant PAH activity analysis method using the cellular bacteria comprising the step of measuring the growth rate of the cultured cellular bacteria.
  2. 제 1항에 있어서, 상기 인간 PAH를 코딩하는 유전자는 서열번호 2의 염기서열을 갖는 hPAH cDNA인 것을 특징으로 하는 방법.The method of claim 1, wherein the gene encoding human PAH is characterized in that the hPAH cDNA having a nucleotide sequence of SEQ ID NO: 2.
  3. 제 1항에 있어서, 상기 페닐케톤뇨증을 야기시키는 돌연변이는 서열번호 1로 표시되는 인간 PAH의 아미노산 서열에서 F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L 및 R408W로 구성된 군에서 선택되는 미스센스 돌연변이(missense mutation)인 것을 특징으로 하는 방법.The method of claim 1, wherein the mutation causing the phenylketonuria is selected from the group consisting of F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W in the amino acid sequence of human PAH represented by SEQ ID NO: 1 And a sense mutation.
  4. 제 3항에 있어서, 상기 미스센스 돌연변이는 서열번호 3 내지 18로 표시되는 프라이머를 이용하여 해당 코돈의 염기가 치환된 것을 특징으로 하는 방법.The method of claim 3, wherein the missense mutation is characterized in that the base of the codon is substituted using a primer represented by SEQ ID NO: 3 to 18.
  5. 제 1항에 있어서, 상기 세포성점균은 딕티오스텔리움 디스코이데움(Dictyostelium discoideum)인 것을 특징으로 하는 방법.The method of claim 1, wherein the cellular slime mold is Dictyostelium discoideum .
  6. 제 1항에 있어서, 상기 세포성점균의 생장 속도는 PAH 활성과 선형상관관계에 있는 것을 특징으로 하는 방법.The method of claim 1, wherein the growth rate of the cellular bacilli is linearly correlated with PAH activity.
  7. 1) 서열번호 1의 아미노산 서열로 표시되는 인간 PAH(phenylalanine hydroxylase)에서 F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L 및 R408W로 구성된 군에서 선택된 아미노산이 치환된 돌연변이 hPAH를 코딩하는 폴리뉴클레오티드가 도입된 재조합벡터로 형질전환된 세포성점균을 제조하는 단계;1) A poly encoding a mutant hPAH substituted with an amino acid selected from the group consisting of F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W in human phenylalanine hydroxylase represented by the amino acid sequence of SEQ ID NO: 1 Preparing a cellular bacterium transformed with the recombinant vector into which the nucleotide is introduced;
    2) 상기 형질전환체된 세포성점균을 PAH에 대한 약리학적 샤프론(pharmacological chaperone) 후보물질이 첨가된 티로신-결핍 최소배양배지에서 배양하는 단계; 및 2) culturing the transformed cellular mycobacteria in a tyrosine-deficient minimal culture medium to which a pharmacological chaperone candidate for PAH has been added; And
    3) 상기 배양 후 세포성점균의 생장 속도가 후보물질 비처리군과 비교하여 증가된 경우, 상기 후보물질을 PAH에 대한 약리학적 샤프론으로 판단하는 단계를 포함하는, 세포성점균을 이용한 페닐케톤뇨증(phenylketonuria) 치료제의 스크리닝 방법.3) when the growth rate of the cellular bacterium after the culture is increased compared to the non-treated group of the candidate substance, the candidate substance is determined as pharmacological saffron for PAH, phenylketonuria using the cellular bacterium ( phenylketonuria) method of screening for therapeutic agents.
  8. 제 7항에 있어서, 상기 1) 단계에서 재조합벡터는 돌연변이 hPAH 유전자가 pDXA-3H 벡터에 삽입된, 도 5에 개시된 재조합벡터인 것을 특징으로 하는, 세포성점균을 이용한 페닐케톤뇨증 치료제의 스크리닝 방법.The method of claim 7, wherein the recombinant vector in step 1) is a recombinant vector disclosed in FIG. 5 in which a mutant hPAH gene is inserted into a pDXA-3H vector. 9.
  9. 제 7항에 있어서, 상기 2) 단계에서 약리학적 샤프론 후보물질은 천연화합물, 합성화합물, 효소, 단백질 또는 핵산인 것을 특징으로 하는, 세포성점균을 이용한 페닐케톤뇨증 치료제의 스크리닝 방법.The method of claim 7, wherein the pharmacological chaperone candidate in step 2) is a natural compound, a synthetic compound, an enzyme, a protein, or a nucleic acid.
  10. 서열번호 1의 아미노산 서열로 표시되는 인간 PAH(phenylalanine hydroxylase)에서 F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L 및 R408W로 구성된 군에서 선택된 아미노산이 치환된 돌연변이 hPAH를 코딩하는 폴리뉴클레오티드가 도입된, 도 5에 개시되어 있는 재조합벡터 pDXA-hPAHs.In the human PAH (phenylalanine hydroxylase) represented by the amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding a mutant hPAH substituted with an amino acid selected from the group consisting of F39L, K42I, L48S, I65T, R252Q, L255V, T278I, S349L and R408W Introduced, the recombinant vector pDXA-hPAHs disclosed in FIG.
  11. 제 10항에 따른 재조합벡터 pDXA-hPAHs로 형질전환된 딕티오스텔리움 디스코이데움(Dictyostelium discoideum) 균주. Dictyostelium discoideum strain transformed with the recombinant vector pDXA-hPAHs according to claim 10.
PCT/KR2015/006854 2015-01-29 2015-07-03 Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds WO2016122058A1 (en)

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