KR20230095726A - Heat Labile Uracil DNA Glycosylase with high capability of preventing Cross-Contamination - Google Patents
Heat Labile Uracil DNA Glycosylase with high capability of preventing Cross-Contamination Download PDFInfo
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Abstract
Description
본 발명은 높은 교차오염 방지 능력을 갖는 열 불안정성 우라실 DNA 글리코실라제(UDG)에 관한 것으로, 구체적으로는 열민감성이 높아 PCR 과정 중에 열에 의해 쉽게 불활성화 되며, 한번 불활성화가 되고 나면 재활성화가 잘 일어 나지 않는 새로운 열민감성 돌연변이 UDG에 관한 것이다.The present invention relates to a heat-labile uracil DNA glycosylase (UDG) having high cross-contamination prevention ability, and specifically, because of its high heat sensitivity, it is easily inactivated by heat during a PCR process, and once inactivated, reactivation is difficult. It is about a new heat-sensitive mutant UDG that does not occur well.
중합효소연쇄반응(PCR, Polymerase Chain Reaction) 기반의 분자진단은 PCR법을 사용하여 특정 DNA를 증폭하여 질병이나 유전자형을 확인하는 검사이다. 중합효소 연쇄반응은 내열성 세균 유래의 DNA 합성효소를 이용하여 시험관 내에서 특정 핵산 부위를 대량으로 증폭하여 유용한 유전자를 분리하거나 확인한다(Erlich, 1989; Shin 등, 2005).Molecular diagnosis based on PCR (Polymerase Chain Reaction) is a test that confirms a disease or genotype by amplifying a specific DNA using the PCR method. Polymerase chain reaction uses DNA synthase derived from heat-resistant bacteria to amplify a specific nucleic acid site in large quantities in vitro to isolate or identify useful genes (Erlich, 1989; Shin et al., 2005).
PCR은 매우 민감하고 신속한 분자 진단 검사법이지만 검출하고자 하는 대상과 PCR에 의해 증폭된 산물이 DNA로 서로 동일하기 때문에 위양성 문제가 발생할 가능성이 높다. 특히, 이전 반응에서 증폭된 PCR 산물이나 검체의 처리 과정 중에 발생된 캐리오버 오염(carry-over contamination)에 의해 위양성 증폭이 일어날 수 있다. 실제로 PCR 진단검사를 실시하는 진단 실험실의 경우 동일한 프라이머를 사용하는 PCR 증폭 실험을 수천에서 수만 회 반복해서 진행하기 때문에, 이전 반응에서 증폭된 PCR 산물이 에러로졸 등의 형태로 공기 중을 떠돌다가 다음번 반응 튜브에 혼입되어 위양성 증폭을 일으킨다는 예는 다양하게 보고되어 오고 있다.PCR is a very sensitive and rapid molecular diagnostic test, but since the target to be detected and the product amplified by PCR are identical to each other as DNA, false positives are likely to occur. In particular, false positive amplification may occur due to carry-over contamination generated during the processing of a PCR product or sample amplified in a previous reaction. In the case of diagnostic laboratories that actually conduct PCR diagnostic tests, PCR amplification experiments using the same primers are repeated thousands to tens of thousands of times, so the PCR products amplified in the previous reaction float in the air in the form of aerosols, etc. Various examples have been reported of mixing in the next reaction tube and causing false positive amplification.
이러한 캐리오버 오염을 해결하기 위해 현재 가장 널리 사용되는 방법은 Uracil DNA Glycosylase를 이용한 방법이다. 우라실-DNA 글리코실라제 (uracil-DNA glycosylase; 이하 "UDG" 라고 함)는 손상된 DNA를 회복시키는 효소로써 DNA의 손상부위를 인식하여 DNA 내의 우라실 염기와 데옥시리보오스 당 (deoxyribose sugar) 사이의 N-글리코시드 결합을 가수분해하여 DNA 내에서 손상된 염기를 제거한다. UDG는 DNA 내의 염기 중에서 우라실 염기만을 특이적으로 제거하는 기질에 대한 특이성이 있다(Lindahl, 1974; Cone 등, 1977).The most widely used method to solve this carryover contamination is the method using Uracil DNA Glycosylase. Uracil-DNA glycosylase (hereafter referred to as "UDG") is an enzyme that repairs damaged DNA and recognizes the damaged site of DNA to form an N between uracil base and deoxyribose sugar in DNA. -Hydrolysis of glycosidic bonds to remove damaged bases in DNA. UDG has specificity for substrates that specifically remove only uracil bases among bases in DNA (Lindahl, 1974; Cone et al., 1977).
UDG 처리법은 먼저, PCR 반응액에 dTTP/dUTP의 혼합물을 첨가하여 PCR을 수행함으로써 T와 U가 혼합된 증폭산물을 만들어낸다. 이후 반응부터는 PCR 반응을 수행할 때 UDG 효소를 PCR 반응액에 첨가하여 37~50℃, 5~30분의 선행 반응을 포함시켜 PCR 진단을 수행한다. 이러한 과정을 통해 Carryover contamination이 발생할 경우 오염된 DNA는 dU를 포함하게 되며, 이들은 UDG에 의해 U base가 제거된다. UDG가 처리되어 베이스가 없어진 DNA는 불안정하기 때문에 PCR 과정 중에 열에 의해 짧은 단편으로 잘려지게 되고, 따라서 이들은 더이상 주형 DNA로 사용되지 못한다.In the UDG treatment method, PCR is first performed by adding a mixture of dTTP/dUTP to the PCR reaction solution, thereby generating an amplification product in which T and U are mixed. From the subsequent reaction, PCR diagnosis is performed by including the preceding reaction at 37 to 50 ° C. for 5 to 30 minutes by adding the UDG enzyme to the PCR reaction solution when performing the PCR reaction. When carryover contamination occurs through this process, the contaminated DNA contains dU, and the U base is removed by UDG. Because the base-free DNA treated with UDG is unstable, it is cut into short fragments by heat during the PCR process, and thus they cannot be used as template DNA any longer.
UDG 처리법은 필요한 시약(UDG 효소, dUTP)을 PCR 반응액에 포함된 채로 사용할 수 있으며 일반적인 PCR 프로그램에 한 step만 추가해주면 되기 때문에 단일 반응/단일 과정으로 캐리오버 오염을 손쉽게 방지할 수 있다는 장점이 있다.The UDG treatment method has the advantage that the necessary reagents (UDG enzyme, dUTP) can be used while being included in the PCR reaction solution, and carryover contamination can be easily prevented with a single reaction/single process because only one step is required to be added to a general PCR program. there is.
그러나 현재 널리 사용되는 UDG 효소는 대부분 대장균에서 유래하여 열적 안정성이 높으며, 심지어 PCR 과정이 종료된 이후에도 활성이 살아있는 경우가 종종 있다. 이 때문에 증폭 산물을 즉시 아가로스젤로 분석하지 않으면 증폭 산물 자체가 분해되는 현상이 발생하는 문제점이 있다.However, most of the currently widely used UDG enzymes are derived from Escherichia coli and have high thermal stability, and are often active even after the PCR process is completed. For this reason, there is a problem in that the amplification product itself is decomposed if the amplification product is not immediately analyzed with an agarose gel.
나아가 복잡한 Multiplex Realtime PCR의 경우 TaqMan 프로브 서열 설계의 한계 때문에 비교적 낮은 온도(약 50 ℃)에서 어닐링(annealing) 반응을 수행해야 하는 경우가 있다. 이때 대장균 유래 UDG 효소의 경우, 해당 온도에서 활성을 보유하고 있으므로 적절하게 증폭된 산물조차 분해하여 전체적인 PCR의 민감도를 저해시키는 (Ct value의 delay) 문제가 발생한다. 이는 PCR 진단 키트의 성능을 하락시키는 원인으로 작용한다.Furthermore, in the case of complex multiplex real-time PCR, there are cases in which an annealing reaction must be performed at a relatively low temperature (about 50 ° C.) due to limitations in designing TaqMan probe sequences. At this time, in the case of E. coli-derived UDG enzyme, since it has activity at the corresponding temperature, even an appropriately amplified product is decomposed, resulting in a problem of inhibiting the overall PCR sensitivity (delay of Ct value). This acts as a cause of deteriorating the performance of the PCR diagnostic kit.
대장균 유래 UDG 이외에 비교적 낮은 온도에서 최적 활성을 보이는 Gadus morhua에서 유래한 UDG가 있다. 그러나 이는 대장균에서 재조합 형태로 발현시키기 때문에 E. coli UDG에 비해 발현율 및 용해도가 낮아 단위 효소 당 정제 비용이 높고 정제 과정이 상대적으로 복잡한 단점이 있다.In addition to UDG derived from Escherichia coli, there is UDG derived from Gadus morhua, which shows optimal activity at a relatively low temperature. However, since it is expressed in a recombinant form in E. coli, the expression rate and solubility are lower than that of E. coli UDG, so the purification cost per unit enzyme is high and the purification process is relatively complicated.
따라서 열민감성이 높아 PCR 등과 같은 반응 과정 중에 열에 의해 쉽게 불활성화되며, 한번 불활성화가 되고 나면 재활성화가 되지 않은 UDG의 개발이 필요하다.Therefore, it is necessary to develop UDG, which is easily inactivated by heat during a reaction process such as PCR due to its high heat sensitivity, and which is not reactivated once inactivated.
본 발명의 목적은 높은 교차오염 방지 능력을 갖는 열 불안정성 우라실 DNA 글리코실라제을 제공하는 데 있다.An object of the present invention is to provide a heat labile uracil DNA glycosylase having high anti-cross-contamination ability.
상기 과제를 해결하기 위하여 본 발명은 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제를 제공한다.In order to solve the above problems, the present invention provides a maltose-binding protein (MBP)-linked uracil DNA glycosylase.
상기 우라실 DNA 글리코실라제는 히스티딘 결합 우라실 DNA 글리코실라제 활성과 비교하여 최소 32배 활성이 증가함으로써 소량의 사용에도 탁월한 교차오염 방지 능력을 가질 수 있다.The activity of the uracil DNA glycosylase is increased at least 32-fold compared to the activity of histidine-linked uracil DNA glycosylase, so that even a small amount can have excellent cross-contamination prevention ability.
상기 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제는 서열번호 3의 염기서열에 의하여 암호화될 수 있다. 또한 상기 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제는 서열번호 4의 아미노산서열을 포함하는 것을 특징으로 한다.Uracil DNA glycosylase bound to the maltose-binding protein (MBP) may be encoded by the nucleotide sequence of SEQ ID NO: 3. In addition, the uracil DNA glycosylase to which the maltose-binding protein (MBP) is bound comprises the amino acid sequence of SEQ ID NO: 4.
본 발명은 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제를 포함하는 PCR(polymerase chain reaction) 조성물을 제공한다. 이때 상기 PCR(polymerase chain reaction)은 중합효소 연쇄반응(Polymerase Chain Reaction, PCR), 역전사 중합효소 연쇄반응 (Reverse Transcription Polymerase Chain Reaction, RT-PCR) 및 등온증폭(isothermal amplification) PCR로 이루어진 군에서 선택된 어느 하나일 수 있다.The present invention provides a polymerase chain reaction (PCR) composition comprising uracil DNA glycosylase to which maltose-binding protein (MBP) is bound. At this time, the polymerase chain reaction (PCR) is selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and isothermal amplification PCR. can be either
본 발명은 또한 서열번호 3으로 표시되는 염기서열을 갖는 우라실-DNA 글리코실라제 폴리뉴클레오티드를 제공한다. 상기 서열번호 3으로 표시되는 염기서열의 폴리뉴클레오티드는 DNA 또는 RNA 일 수 있다. 또한 상기 폴리뉴클레오티드는 자연에서 분리되거나 또는 화학적 합성법에 의해 제조될 수 있다.The present invention also provides a uracil-DNA glycosylase polynucleotide having the nucleotide sequence represented by SEQ ID NO: 3. The polynucleotide of the nucleotide sequence represented by SEQ ID NO: 3 may be DNA or RNA. In addition, the polynucleotide may be isolated from nature or prepared by chemical synthesis.
상기 본 발명에 따른 우라실-DNA 글리코실라제를 암호화하는 폴리뉴클레오티드는 적합한 발현 벡터내로 삽입되어 숙주세포를 형질전환할 수 있다. 이때 벡터는 UDG 단백질을 코딩하는 폴리뉴클레오티드 서열이 삽입 또는 도입될 수 있는 당분야에 공지된 플라스미드, 바이러스 또는 기타 매개체를 의미한다. 본 발명에 따른 폴리뉴클레오티드 서열은 발현 조절 서열에 작동 가능하게 연결될 수 있으며, 상기 작동 가능하게 연결된 유전자 서열과 발현 조절 서열은 선택 마커 및 복제 개시점(replication origin)을 같이 포함하고 있는 하나의 벡터 내에 포함될 수 있다.The polynucleotide encoding the uracil-DNA glycosylase according to the present invention can be inserted into a suitable expression vector to transform a host cell. In this case, the vector refers to a plasmid, virus, or other medium known in the art into which a polynucleotide sequence encoding a UDG protein can be inserted or introduced. The polynucleotide sequence according to the present invention can be operably linked to an expression control sequence, and the operably linked gene sequence and expression control sequence are contained within a single vector containing a selectable marker and a replication origin. can be included
작동 가능하게 연결(operably linked)된다는 것은 적절한 분자가 발현 조절 서열에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 발현 조절 서열일 수 있다. 상기 발현 조절 서열(expression control sequence)이란 특정한 숙주 세포에서 작동 가능하게 연결된 폴리뉴클레오티드 서열의 발현을 조절하는 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 실시하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 포함한다. 상기 플라스미드의 예로는 대장균 유래 플라스미드(pBR322, pBR325, pUC118 및 pUC119), 바실러스 서브틸러스 유래 플라스미드(pUB110 및 pTP5) 및 효모 유래 플라스미드(YEp13, YEp24 및 YCp50) 등이 있으며 상기 바이러스는 레트로바이러스, 아데노바이러스 또는 백시니아 바이러스와 같은 동물 바이러스, 배큘로바이러스와 같은 곤충 바이러스가 사용될 수 있으나 이에 제한되는 것은 아니며, 당업자라면 본 발명에 따른 폴리뉴클레오티드를 숙주세포에 도입시키는데 적합한 벡터를 사용할 수 있으며, 바람직하게는 단백질 발현 유도 및 발현된 단백질의 분리가 용이하도록 디자인된 벡터를 사용할 수 있다.Operably linked can be genes and expression control sequences linked in such a way as to enable gene expression when appropriate molecules are linked to the expression control sequences. The expression control sequence refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include promoters to effect transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences to control termination of transcription and translation. Examples of the plasmids include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119), Bacillus subtilus-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50), and the viruses include retroviruses, adenoviruses, and adenoviruses. Viruses, animal viruses such as vaccinia virus, and insect viruses such as baculovirus may be used, but are not limited thereto, and those skilled in the art may use vectors suitable for introducing the polynucleotide according to the present invention into host cells, preferably. may use a vector designed to facilitate protein expression induction and isolation of the expressed protein.
본 발명은 서열번호 3으로 표시되는 염기서열을 갖는 우라실-DNA 글리코실라제 폴리뉴클레오티드 및 상기 폴리뉴클레오티드를 포함하는 재조합 벡터를 제공한다. 또한 상기 벡터로 형질전환된 세균을 제공한다. 상기 형질전환된 세균은 대량배양되어 본 발명의 우라실-DNA 글리코실라제를 대량 생산할 수 있다.The present invention provides a uracil-DNA glycosylase polynucleotide having the nucleotide sequence represented by SEQ ID NO: 3 and a recombinant vector containing the polynucleotide. In addition, bacteria transformed with the vector are provided. The transformed bacteria can be cultured in large quantities to mass-produce the uracil-DNA glycosylase of the present invention.
본 발명은 열번호 3으로 표시되는 염기서열을 갖는 우라실-DNA 글리코실라제 폴리뉴클레오티드를 포함하는 벡터로 형질전환된 세균을 대량 배양하여 상기 서열번호 3으로 표시되는 염기서열을 갖는 우라실-DNA 글리코실라제를 생산하는 방법을 제공한다.In the present invention, bacteria transformed with a vector containing the uracil-DNA glycosylase polynucleotide having the nucleotide sequence represented by SEQ ID NO: 3 are mass-cultivated to obtain uracil-DNA glycosylase having the nucleotide sequence represented by SEQ ID NO: 3. Provides a method for producing the agent.
본 발명은 소량의 사용에도 높은 교차오염 방지능력을 가져 시료 핵산의 오염에 의한 위양성으로부터 안전한 PCR검사를 수행할 수 있는 열 불안정성 DNA 할 수 있다.The present invention has a high cross-contamination prevention ability even when used in a small amount, and can perform a PCR test safe from false positives due to contamination of sample nucleic acids.
도 1은 대서양대구(Gadus morhua) UDG의 핵산서열 (서열번호 1)을 나타낸 것이다.
도 2는 대서양대구(Gadus morhua) UDG의 아미노산 서열 (서열번호 2)을 나타낸 것이다.
도 3은 본 발명에 따른 MBP-UDG의 핵산 서열 (서열번호 3)을 나타낸 것이다.
도 4는 본 발명에 따른 MBP-UDG의 아미노산 서열 (서열번호 4)을 나타낸 것이다.
도 5는 본 발명에 따른 UDG 클로닝을 위하여 제작된 플라스미드의 구조를 도시한 것이다. A. pET-GUH, B. pMAL-GU
도 6은 본 발명에 따른 UDG가 삽입된 플라스미드에 의하여 형질전환된 BL21 cell lysate의 SDS-PAGE 사진이다. A. BL21/pET-GUH cell lysate(12% SDS-PAGE) [1: BL21 cell lysate(Soluble fraction), 2: BL21 cell lysate(Insoluble fraction), 3~8: BL21/pET-GUH cell lysate(Soluble fraction, Insoluble fraction), 3, 4: 1mM IPTG induction, 5, 6: 0.1mM IPTG induction, 7, 8: no induction, C: control(E.coli UDG)], B. BL21/pMAL-GU cell lysate(12% SDS-PAGE) [1: BL21 cell lysate(Soluble fraction), 2: BL21 cell lysate(Insoluble fraction), 3~6: BL21/pMAL-GU cell lysate(Soluble fraction, Insoluble fraction), 3, 4: 0.1mM IPTG induction, 5, 6: 1mM IPTG induction, C: control(E.coli UDG)]
도 7은 BL21/pET-GUH 및 BL21/pMAL-GU로부터 분리, 농축된 열 불안정성 UDG의 SDS-PAGE 사진이다. A. Ni-affinity chromatography를 통한 BL21/pET-GUH로부터 분리된 His tag된 Heat-labile UDG [1: 1: BL21 cell lysate, 2: BL21/pET-GUH whole cell lysate, 3: Ni-affinity loading fraction, 4: Ni-affinity loading flow through fraction, 5: Ni-affinity wash flow through fraction, 6: Ni-affinity Elution fraction], B. MBP-affinity chromatography를 통한 BL21/pMAL로부터 분리된 MBP tag된 Heat-labile UDG [1: BL21/pMAL-GU cell lysate(soluble fraction/MBP-affinity loading sample), 2: Loading flow through fraction, 3: Wash flow through fraction, 4: Elution fraction]
도 8은 His tag된 Heat-labile UDG(GUH), MBP tag된 Heat-labile UDG(M-GU) 및 UDG가 첨가되지 않은 RT-PCR premix(SuPrimeScript RT-PCR premix(SR-7000))의 PCR 증폭된 밴드를 나타내는 사진이다.
도 9는 His tag된 Heat-labile UDG(GUH), MBP tag된 Heat-labile UDG(M-GU)를 이용한 2nd RT-PCR 시, UDG 활성 비교한 나타내는 사진이다.
도 10은 본 발명에 따른 MBP tag된 Heat-labile UDG를 포함(UDG(+), 빨간색) 또는 포함하지 않는(UDG(-), 검정색) 1st real-time RT-PCR 또는 2nd real-time RT-PCR 결과를 나타내는 그래프이다.1 is Atlantic cod ( Gadus morhua ) UDG nucleic acid sequence (SEQ ID NO: 1).
2 is Atlantic cod ( Gadus morhua ) shows the amino acid sequence of UDG (SEQ ID NO: 2).
Figure 3 shows the nucleic acid sequence (SEQ ID NO: 3) of MBP-UDG according to the present invention.
Figure 4 shows the amino acid sequence (SEQ ID NO: 4) of MBP-UDG according to the present invention.
Figure 5 shows the structure of a plasmid constructed for UDG cloning according to the present invention. A. pET-GUH, B. pMAL-GU
6 is an SDS-PAGE photograph of BL21 cell lysate transformed with the plasmid into which UDG according to the present invention is inserted. A. BL21/pET-GUH cell lysate (12% SDS-PAGE) [1: BL21 cell lysate (Soluble fraction), 2: BL21 cell lysate (Insoluble fraction), 3~8: BL21/pET-GUH cell lysate (Soluble fraction) fraction, insoluble fraction), 3, 4: 1 mM IPTG induction, 5, 6: 0.1 mM IPTG induction, 7, 8: no induction, C: control (E.coli UDG)], B. BL21/pMAL-GU cell lysate (12% SDS-PAGE) [1: BL21 cell lysate (Soluble fraction), 2: BL21 cell lysate (Insoluble fraction), 3~6: BL21/pMAL-GU cell lysate (Soluble fraction, Insoluble fraction), 3, 4 : 0.1mM IPTG induction, 5, 6: 1mM IPTG induction, C: control (E.coli UDG)]
7 is an SDS-PAGE image of heat labile UDG isolated and concentrated from BL21/pET-GUH and BL21/pMAL-GU. A. His-tagged heat-labile UDG isolated from BL21/pET-GUH through Ni-affinity chromatography [1: 1: BL21 cell lysate, 2: BL21/pET-GUH whole cell lysate, 3: Ni-affinity loading fraction , 4: Ni-affinity loading flow through fraction, 5: Ni-affinity wash flow through fraction, 6: Ni-affinity elution fraction], B. MBP-tagged heat-labile isolated from BL21/pMAL through MBP-affinity chromatography UDG [1: BL21/pMAL-GU cell lysate (soluble fraction/MBP-affinity loading sample), 2: Loading flow through fraction, 3: Wash flow through fraction, 4: Elution fraction]
8 is PCR of His-tagged Heat-labile UDG (GUH), MBP-tagged Heat-labile UDG (M-GU), and RT-PCR premix without UDG (SuPrimeScript RT-PCR premix (SR-7000)) This is a picture showing the amplified band.
9 is a photograph showing comparison of UDG activity in 2 nd RT-PCR using His-tagged heat-labile UDG (GUH) and MBP-tagged heat-labile UDG (M-GU).
10
본 발명은 높은 교차오염 방지 능력을 갖는 열 불안정성 우라실 DNA 글리코실라제에 관한 것이다. Uracil-DNA 글리코실라제(UDG, EC 3.2.2.3)는 단일 또는 이중 가닥 DNA에서 promutagenic 우라실 잔기의 가수분해를 촉매하여 유리 우라실 및 무염기 DNA를 생성하는 DNA 복구 단백질이다. 특히 대서양대구(Gadus morhua) UDG는 저온 적응종에서 유래하여 열민감성이 높아 PCR 과정 중에 열에 의해 쉽게 불활성화되며, 한번 불활성화가 되고나면 재활성화가 잘 일어나지 않는다.The present invention relates to a heat labile uracil DNA glycosylase having high anti-cross-contamination ability. Uracil-DNA glycosylase (UDG, EC 3.2.2.3) is a DNA repair protein that catalyzes the hydrolysis of promutagenic uracil residues in single- or double-stranded DNA to produce free uracil and free DNA. Especially Atlantic cod ( Gadus) morhua ) UDG is derived from a low-temperature adaptable species and has high heat sensitivity, so it is easily inactivated by heat during the PCR process, and once inactivated, reactivation does not occur well.
한편, 말토스 결합 단백질(maltose-binding protein, MBP)은 재조합 단백질에 연결하여 재조합 단백질과 함께 발현시키는 경우, 말토스 결합 비드 또는 아가로스에 결합되므로, 목적한 재조합 단백질의 분리 정제가 용이해진다. 본 발명자는 대서양대구(Gadus morhua) UDG의 UDG 활성을 높이기 위한 여러 돌연변이를 연구하는 과정에서 서열번호 1의 열 불안정성 UDG을 수득하였으며, 특히 이를 MBP와 결합하여 발현시키는 경우, UDG 활성 자체가 크게 증가하는 것을 확인하고 본 발명을 완성하였다.On the other hand, when maltose-binding protein (MBP) is linked to a recombinant protein and expressed together with the recombinant protein, it binds to maltose-binding beads or agarose, thereby facilitating separation and purification of the desired recombinant protein. The present inventors obtained heat-labile UDG of SEQ ID NO: 1 in the process of studying several mutations to increase UDG activity of Atlantic cod ( Gadus morhua ) UDG, and in particular, when combined with MBP and expressed, UDG activity itself greatly increased It was confirmed that the present invention was completed.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.
<< 실시예Example 1. 열 불안정성 1. Thermal instability UDGUDG 유전자의 코돈 최적화 및 codon optimization of genes and 클로닝cloning Heat labile Heat labile UDGUDG gene(codon optimization) cloning> gene (codon optimization) cloning>
대서양대구(Gadus morhua) UDG (서열번호 1) 를 E. coli에서 발현시키기 위해 codon optimization 진행 후, signal sequence부위를 제외한 UDG DNA sequence(종결코돈 제외-663bp)를 합성하였다. 상기 대서양대구(Gadus morhua) UDG의 아미노산 서열은 서열번호 2에 나타내었다. 도 1은 대서양대구(Gadus morhua) UDG의 핵산서열 (서열번호 1)을, 도 2는 대서양대구(Gadus morhua) UDG의 아미노산 서열 (서열번호 2)을 나타낸 것이다. 그리고 도 3은 본 발명에 따른 MBP-UDG의 핵산 서열 (서열번호 3)을, 도 4는 본 발명에 따른 MBP-UDG의 아미노산 서열 (서열번호 4)을 나타낸 것이다. Atlantic Cod ( Gadus) morhua ) UDG (SEQ ID NO: 1) was synthesized after codon optimization was performed to express UDG (SEQ ID NO: 1) in E. coli, and UDG DNA sequence (excluding stop codon -663bp) excluding the signal sequence region was synthesized. The Atlantic cod ( Gadus morhua ) The amino acid sequence of UDG is shown in SEQ ID NO: 2. 1 is Atlantic cod ( Gadus morhua ) UDG nucleic acid sequence (SEQ ID NO: 1), Figure 2 is Atlantic cod ( Gadus morhua ) shows the amino acid sequence of UDG (SEQ ID NO: 2). 3 shows the nucleic acid sequence (SEQ ID NO: 3) of MBP-UDG according to the present invention, and FIG. 4 shows the amino acid sequence (SEQ ID NO: 4) of MBP-UDG according to the present invention.
상기 합성한 유전자를 pET-vector system의 His-tag을 사용할 수 있도록 클로닝하였다(도 5A). 이어 UDG gene의 active form 발현을 증대시키기 위해 MBP(Maltose-binding protein) binding domain gene을 함께 클로닝하였다.(도 5B). 각각 클로닝된 플라스미드는 발현용 E.coli host cell BL21에 transformation하여 BL21/pET-GUH 및 BL21/pMAL-GU를 제작하였다. 제작된 재조합 플라스미드를 도 5에 나타내었다.The synthesized gene was cloned so that the His-tag of the pET-vector system could be used (FIG. 5A). Subsequently, in order to increase the expression of the active form of the UDG gene, the MBP (Maltose-binding protein) binding domain gene was cloned together (FIG. 5B). Each cloned plasmid was transformed into E.coli host cell BL21 for expression, and BL21/pET-GUH and BL21/pMAL-GU were prepared. The constructed recombinant plasmid is shown in FIG. 5 .
이때 본 발명에 따른 MBP-UDG의 핵산 서열은 서열번호 3에, MBP-UDG의 아미노산 서열은 서열번호 4에 나타내었다.At this time, the nucleic acid sequence of MBP-UDG according to the present invention is shown in SEQ ID NO: 3, and the amino acid sequence of MBP-UDG is shown in SEQ ID NO: 4.
<< 실시예Example 2. 열 불안정성 2. Thermal instability UDGUDG 유전자의 발현 및 발효 Heat-labile Gene expression and fermentation Heat-labile UDGUDG expression fermentation> expression fermentation>
BL21/pET-GUH 및 BL21/pMAL-GU를 배양하여 cell을 파쇄 후 SDS-PAGE분석을 통하여 UDG 발현을 확인하였다(도 6). 도 6에서 보는 바와 같이, BL21/pET-GUH의 경우 대부분 insoluble form(lane 4, 6)으로 발현되었고, BL21/pMAL-GU의 경우 soluble form(lane 3, 5)으로 대부분 발현되었다.BL21/pET-GUH and BL21/pMAL-GU were cultured, the cells were disrupted, and UDG expression was confirmed through SDS-PAGE analysis (FIG. 6). As shown in FIG. 6 , BL21/pET-GUH was mostly expressed in insoluble form (
<< 실시예Example 3. 열 불안정성 3. Thermal instability UDGUDG 분리 Heat-labile Separation Heat-labile UDGUDG Purification( Purification ( NiNi -affinity chromatography, -affinity chromatography, MBPMBP -affinity chromatography)>-affinity chromatography)>
배양된 BL21/pET-GUH 및 BL21/pMAL-GU로부터 열 불안정성 UDG를 분리 정제하였다. 배양액 BL21/pET-GUH 배양액을 4000rpm에서 10분간 원심분리하여 배양상등액을 제거하고, lysis buffer에 cell을 재현탁하였다. Cell 현탁액 비커를 아이스가 담긴 아이스박스에 넣고, 초음파 파쇄기로 cell을 파쇄하였다.Heat labile UDG was isolated and purified from cultured BL21/pET-GUH and BL21/pMAL-GU. Culture medium The BL21/pET-GUH culture medium was centrifuged at 4000 rpm for 10 minutes to remove the culture supernatant, and the cells were resuspended in lysis buffer. The cell suspension beaker was placed in an ice box containing ice, and the cells were disrupted with an ultrasonic disruptor.
BL21/pET-GUH의 6xHis tag을 이용한 affinity chromatography를 위해 Ni-agarose resin을 column에 충진하였다. Column을 equilibration buffer로 평형화 시키고, BL21/pET-GUH 조효소액을 흘려주었다. 이후, Column volume의 5배의 washing buffer를 흘려 결합이 일어나지 않은 단백질을 제거하고, 250mM imidazole을 포함한 buffer를 흘려 column에서 UDG를 elution하였다. Elution된 UDG solution은 투석을 통해 storage buffer로 buffer를 바꾸어 주었다. 각 단계에서의 solution을 sampling해서 SDS-PAGE를 통해 정제 효율을 분석하였다. His tag을 이용한 affinity purification을 거친 샘플에서 정제, 농축된 UDG는 SDS-PAGE를 통해 확인하여 도 7A에 나타내었다.Ni-agarose resin was filled in the column for affinity chromatography using the 6xHis tag of BL21/pET-GUH. The column was equilibrated with equilibration buffer, and BL21/pET-GUH crude enzyme solution was flowed. Thereafter,
도 7A에서 보는 바와 같이, Ni-affinity chromatography를 통하여 BL21/pET-GUH로부터 분리된 His tag된 Heat-labile UDG 은 30kD 부근에서 강한 밴드를 나타내어 분리, 농축된 것을 확인하였다.As shown in FIG. 7A, His-tagged heat-labile UDG isolated from BL21/pET-GUH through Ni-affinity chromatography showed a strong band around 30 kD, confirming that it was separated and concentrated.
BL21/pMAL-GU로부터 MBP tag된 열 불안정성 UDG를 분리 정제는 상기 BL21/pET-GUH로부터 His tag된 열 불안정성 UDG를 분리 정제에서 Ni-affinity chromatography를 MBP-affinity chromatography로 대체하는 것을 제외하고는 동일한 방법으로 수행하였다. BL21/pMAL-GU의 MBP tag을 이용한 affinity chromatography를 위해 MBP-agarose resin을 column에 충진하였다. Column을 wash buffer로 평형화시키고, BL21/pMAL-GU 조효소액을 흘려주었다. Column volume의 5배의 washing buffer를 흘려 결합이 일어나지 않은 단백질을 제거하였다. 10mM maltose을 포함한 buffer를 흘려 column에서 UDG를 elution하였다. Elution된 UDG solution에서 투석을 통해 storage buffer로 buffer를 바꾸어 주었다. 각 단계에서의 solution을 sampling해서 SDS-PAGE를 통해 정제 효율을 분석하였다. MBP tag을 이용한 affinity purification을 거친 샘플에서 UDG가 정제, 농축된 UDG는 SDS-PAGE를 통해 확인하여 도 7B에 나타내었다.The separation and purification of MBP-tagged heat-labile UDG from BL21/pMAL-GU was the same except that Ni-affinity chromatography was replaced with MBP-affinity chromatography in the separation and purification of His-tagged heat-labile UDG from BL21/pET-GUH. method was carried out. For affinity chromatography using the MBP tag of BL21/pMAL-GU, the column was filled with MBP-agarose resin. The column was equilibrated with wash buffer, and BL21/pMAL-GU crude enzyme solution was flowed. Unbound proteins were removed by flowing
도 7B에서 보는 바와 같이, MBP-affinity chromatography를 통하여 BL21/pMAL-GU로부터 분리된 MBP tag된 Heat-labile UDG 은 70kD 부근에서 강한 밴드를 나타내어 분리, 농축된 것을 확인하였다. As shown in FIG. 7B, MBP-tagged heat-labile UDG isolated from BL21/pMAL-GU through MBP-affinity chromatography showed a strong band around 70 kD, confirming that it was separated and concentrated.
<< 실시예Example 4. Heat labile 4. Heat labile UDGUDG 첨가 RT- Added RT- PCRPCR premix 제조 및 성능 확인> Confirmation of premix fabrication and performance>
His tag된 Heat-labile UDG 및 MBP tag된 Heat-labile UDG를 사용하여 각각 RT-PCR premix(SuPrimeScript RT-PCR premix with UDG(USR-7000)) 제조하고, UDG가 첨가되지 않은 RT-PCR premix(SuPrimeScript RT-PCR premix(SR-7000))와 비교하였다. Human lung RNA(10ng/㎕, 1ng/㎕, 100pg/㎕, 10pg/㎕, Negative)를 template로 사용하고, GAPDH gene을 증폭할 수 있는 primer를 사용하여 617bp, 354bp, 215bp의 단편을 RT-PCR을 수행하여 그 결과를 도 8에 나타내었다.RT-PCR premix (SuPrimeScript RT-PCR premix with UDG (USR-7000)) was prepared using His-tagged Heat-labile UDG and MBP-tagged Heat-labile UDG, respectively, and RT-PCR premix without UDG ( It was compared with SuPrimeScript RT-PCR premix (SR-7000)). Human lung RNA (10ng/μl, 1ng/μl, 100pg/μl, 10pg/μl, Negative) was used as a template and 617bp, 354bp, and 215bp fragments were RT-PCR using primers capable of amplifying the GAPDH gene. was performed and the results are shown in FIG. 8 .
도 8에서 보는 바와 같이, His tag된 Heat-labile UDG(GUH) 및 MBP tag된 Heat-labile UDG(M-GU)가 포함된 RT-PCR premix(SuPrimeScript RT-PCR premix with UDG(USR-7000)) 사용하는 경우에도 UDG가 첨가되지 않은 RT-PCR premix(SuPrimeScript RT-PCR premix(SR-7000))와 비교하여 증폭된 밴드에 영향을 주지 않았다.As shown in FIG. 8, RT-PCR premix (SuPrimeScript RT-PCR premix with UDG (USR-7000)) containing His-tagged Heat-labile UDG (GUH) and MBP-tagged Heat-labile UDG (M-GU) ) did not affect the amplified band compared to the RT-PCR premix (SuPrimeScript RT-PCR premix (SR-7000)) to which UDG was not added.
<< 실시예Example 5. 5. MBPMBP tag된tagged Heat-labile Heat-labile UDGUDG 성능 확인> Performance check>
His tag된 Heat-labile UDG 및 MBP tag된 Heat-labile UDG를 추가한RT-PCR premix(SuPrimeScript RT-PCR premix with UDG(USR-7000))의 UDG 활성을 확인하였다. Human RNA를 template로 GAPDH gene을 증폭하면서 BL21/pMAL-GU로부터 정제된 His tag된 Heat-labile UDG 및 BL21/pET-GUH로부터 정제된 MBP tag된 Heat-labile UDG를 각각 1, 1/2, 1/4, 1/8, 1/16, 1/32 unit 혼합하여 표 1과 같은 조건으로 진행하였다. 1차 RT-PCR을 진행하고, PCR Product(1차 RT-PCR)를 이용하여 2차 RT-PCR을 진행하여 그 결과를 도 9에 나타내었다.The UDG activity of RT-PCR premix (SuPrimeScript RT-PCR premix with UDG (USR-7000)) to which His-tagged Heat-labile UDG and MBP-tagged Heat-labile UDG were added was confirmed. While amplifying the GAPDH gene with human RNA template, His-tagged Heat-labile UDG purified from BL21/pMAL-GU and MBP-tagged Heat-labile UDG purified from BL21/pET-GUH were 1, 1/2, 1, respectively. /4, 1/8, 1/16, 1/32 units were mixed and proceeded under the conditions shown in Table 1. The first RT-PCR was performed, and the second RT-PCR was performed using the PCR Product (first RT-PCR), and the results are shown in FIG. 9 .
18 min
56 sec2 hours
18min
56 sec
Conventional
PCR Machine BIOER
Conventional
PCR Machine
도 9에서 보는 바와 같이, UDG가 첨가 되지 않은 RT-PCR premix(SuPrimeScript RT-PCR premix(SR-7000))를 사용한 경우에서는 증폭된 밴드가 확인되었으며(Lane2, 4), UDG가 추가된 RT-PCR premix(SuPrimeScript RT-PCR premix with UDG(USR-7000))를 사용한 경우, UDG의 활성으로 첫번째 1차 RT-PCR에서 증폭된 RT-PCR 산물을 분해하여 증폭을 억제함으로써 교차 오염을 방지하는 것을 확인하였다. 이때 MBP tag된 Heat-labile UDG는 His tag된 Heat-labile UDG보다 높은 UDG 활성을 나타내어, MBP tag된 Heat-labile UDG 1/32 unit의 추가에도 1차 RT-PCR 산물을 거의 완전히 분해한 것을 확인하였다. 이를 통하여 일반적인 His tag된 Heat-labile UDG와 비교하여 매우 높은 UDG 활성을 띠는 것을 확인하였다.As shown in FIG. 9, when the RT-PCR premix (SuPrimeScript RT-PCR premix (SR-7000)) without UDG was used, amplified bands were confirmed (Lane2, 4), and RT-PCR with UDG added When PCR premix (SuPrimeScript RT-PCR premix with UDG (USR-7000)) is used, the activity of UDG degrades the RT-PCR product amplified in the first round of RT-PCR to inhibit amplification, thereby preventing cross-contamination. Confirmed. At this time, MBP-tagged heat-labile UDG showed higher UDG activity than His-tagged heat-labile UDG, confirming that the first RT-PCR product was almost completely degraded even with the addition of 1/32 unit of MBP-tagged heat-labile UDG. did Through this, it was confirmed that UDG activity was very high compared to general His-tagged heat-labile UDG.
<< 실시예Example 6. 6. MBPMBP tag된tagged Heat-labile Heat-labile UDGUDG 첨가 adding qRTqRT -- PCRPCR premix 제조 및 성능 확인> Confirmation of premix fabrication and performance>
상기 실시예 5에서 UDG 활성이 매우 높은 본 발명의 MBP tag된 Heat-labile UDG를 사용하여 real-time RT-PCR premix(SuPrimeScript qRT-PCR Kit with UDG(UQ-5000)) 제조하고 UDG가 첨가되지 않은 real-time RT-PCR premix(SuPrimeScript qRT-PCR Kit(Q-5000))와 비교하였다. Human lung RNA(10ng/㎕, 1ng/㎕, 100pg/㎕, Negative)를 template로 Actin[Cy5], HPRT[FAM], HBS1L[VIC] primer[probe]를 사용하여 real-time PCR을 수행하고 그 결과를 도 10에 나타내었다. UDG 활성 분석 qRT-PCR 조건은 표 2와 같았다.In Example 5, a real-time RT-PCR premix (SuPrimeScript qRT-PCR Kit with UDG (UQ-5000)) was prepared using the MBP-tagged heat-labile UDG of the present invention, which has a very high UDG activity, and UDG was not added. compared with real-time RT-PCR premix (SuPrimeScript qRT-PCR Kit (Q-5000)). Real-time PCR was performed using human lung RNA (10ng/μl, 1ng/μl, 100pg/μl, Negative) as a template using Actin [Cy5], HPRT [FAM], and HBS1L [VIC] primer [probe]. Results are shown in FIG. 10 . UDG activity assay qRT-PCR conditions are shown in Table 2.
20 min
53 sec1 hour
20min
53 sec
도 10에서 보는 바와 같이, 첫번째 real-time RT-PCR을 수행한 경우, MBP tag된 Heat-labile UDG 첨가 유무에 상관없이 첫 번째 real-time RT-PCR 반응에서 동일한 real-time RT-PCR 성능을 나타내었다. 그러나 2차 real-time RT-PCR을 수행하면, Actin[Cy5], HPRT[FAM], HBS1L[VIC] primer[probe]를 사용하여 real-time RT-PCR 수행한 모두에서 본 발명의 MBP tag된 Heat-labile UDG를 사용한 경우, template가 완전히 분해되었음을 확인하였다(빨간색). 즉, UDG가 첨가되지 않은 real-time RT-PCR premix(SuPrimeScript qRT-PCR Kit(Q-5000))를 사용한 경우에서는 증폭된 그래프를 확인할 수 있었으며(검정색), 본 발명에 따른 MBP tag된 Heat-labile UDG 추가된 real-time RT-PCR premix(SuPrimeScript qRT-PCR Kit with UDG(UQ-5000))를 사용한 경우, UDG의 활성으로 첫번째 real-time RT-PCR 산물이 모두 분해되어 real-time RT-PCR 증폭이 되지 않음을 확인하였다.As shown in FIG. 10, when the first real-time RT-PCR was performed, the same real-time RT-PCR performance was obtained in the first real-time RT-PCR reaction regardless of whether or not MBP-tagged heat-labile UDG was added. showed up However, when performing the secondary real-time RT-PCR, the MBP-tagged MBP of the present invention was performed in all of the real-time RT-PCR using Actin [Cy5], HPRT [FAM], and HBS1L [VIC] primer [probe]. When heat-labile UDG was used, it was confirmed that the template was completely degraded (red). That is, in the case of using the real-time RT-PCR premix (SuPrimeScript qRT-PCR Kit (Q-5000)) to which UDG was not added, the amplified graph was confirmed (black), and the MBP-tagged Heat- When using labile UDG-added real-time RT-PCR premix (SuPrimeScript qRT-PCR Kit with UDG (UQ-5000)), the activity of UDG degrades all of the first real-time RT-PCR products, resulting in real-time RT-PCR. It was confirmed that PCR amplification was not performed.
이를 통하여 본 발명의 MBP tag된 Heat-labile UDG는 종래의 His tag된 Heat-labile UDG보다 UDG 활성이 32배 이상 높아 real-time RT-PCR을 포함하는 PCR 수행시, PCR 반복에 의한 template의 교차오염을 효과적으로 방지하는 것을 확인하였다.Through this, the MBP-tagged heat-labile UDG of the present invention has 32 times higher UDG activity than the conventional His-tagged heat-labile UDG, and thus, when performing PCR including real-time RT-PCR, template crossover by PCR repetition It was confirmed that contamination was effectively prevented.
Claims (10)
상기 우라실 DNA 글리코실라제는 히스티딘 결합 우라실 DNA 글리코실라제 활성과 비교하여 최소 32배 활성이 증가한 것을 특징으로 하는 우라실 DNA 글리코실라제.According to claim 1,
The uracil DNA glycosylase is uracil DNA glycosylase, characterized in that the activity is increased by at least 32 times compared to the activity of histidine-linked uracil DNA glycosylase.
상기 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제는 서열번호 3의 염기서열에 의하여 암호화되는 것을 특징으로 하는 우라실 DNA 글리코실라제.According to claim 2,
Uracil DNA glycosylase to which the maltose-binding protein (MBP) is bound is encoded by the nucleotide sequence of SEQ ID NO: 3.
상기 말토스 결합 단백질(maltose-binding protein, MBP)이 결합된 우라실 DNA 글리코실라제는 서열번호 4의 아미노산서열을 포함하는 것을 특징으로 하는 우라실 DNA 글리코실라제.According to claim 2,
Uracil DNA glycosylase to which the maltose-binding protein (MBP) is bound comprises the amino acid sequence of SEQ ID NO: 4.
상기 PCR(polymerase chain reaction)은 중합효소 연쇄반응(Polymerase Chain Reaction, PCR), 역전사 중합효소 연쇄반응 (Reverse Transcription Polymerase Chain Reaction, RT-PCR) 및 등온증폭(isothermal amplification) PCR로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 PCR(polymerase chain reaction) 조성물.According to claim 5,
The polymerase chain reaction (PCR) is any one selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and isothermal amplification PCR. PCR (polymerase chain reaction) composition, characterized in that one.
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