KR101624703B1 - Novel Polypeptides Binding with red fluorescent protein - Google Patents
Novel Polypeptides Binding with red fluorescent protein Download PDFInfo
- Publication number
- KR101624703B1 KR101624703B1 KR1020140098834A KR20140098834A KR101624703B1 KR 101624703 B1 KR101624703 B1 KR 101624703B1 KR 1020140098834 A KR1020140098834 A KR 1020140098834A KR 20140098834 A KR20140098834 A KR 20140098834A KR 101624703 B1 KR101624703 B1 KR 101624703B1
- Authority
- KR
- South Korea
- Prior art keywords
- protein
- fluorescent protein
- red fluorescent
- polypeptide
- leu
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Abstract
본 발명은 적색 형광단백질과 결합할 수 있는 신규한 폴리펩타이드에 관한 것으로, 보다 구체적으로, 적색 형광단백질과 선택적으로 결합하여 생체내 단백질의 움직임을 파악할 수 있는 항체 즉, 폴리펩타이드, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 재조합 벡터, 상기 재조합 벡터가 도입되어 있는 재조합 미생물 및 상기 재조합 미생물을 이용하여 상기 폴리펩타이드를 생산하는 방법에 관한 것이다.
본 발명의 폴리펩타이드는 적색 형광단백질에 대한 결합력이 우수하므로, 세포생물학, 분자생물학뿐만 아니라 기초 의과학 연구 분야에 효과적으로 활용될 수 있다. The present invention relates to a novel polypeptide capable of binding to a red fluorescent protein. More specifically, the present invention relates to an antibody capable of detecting the movement of a protein in vivo selectively binding with a red fluorescent protein, that is, a polypeptide, A polynucleotide encoding the polynucleotide, a recombinant vector containing the polynucleotide, a recombinant microorganism into which the recombinant vector is introduced, and a method of producing the polypeptide using the recombinant microorganism.
Since the polypeptide of the present invention is excellent in binding ability to red fluorescent protein, it can be effectively used in basic biochemical research as well as cell biology and molecular biology.
Description
본 발명은 적색 형광단백질과 결합할 수 있는 신규한 폴리펩타이드에 관한 것으로, 보다 구체적으로, 적색 형광단백질과 선택적으로 결합하여 생체내 단백질의 움직임을 파악할 수 있는 항체 즉, 폴리펩타이드, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 재조합 벡터, 상기 재조합 벡터가 도입되어 있는 재조합 미생물 및 상기 재조합 미생물을 이용하여 상기 폴리펩타이드를 생산하는 방법에 관한 것이다.
The present invention relates to a novel polypeptide capable of binding to a red fluorescent protein. More specifically, the present invention relates to an antibody capable of detecting the movement of a protein in vivo selectively binding with a red fluorescent protein, that is, a polypeptide, A polynucleotide encoding the polynucleotide, a recombinant vector containing the polynucleotide, a recombinant microorganism into which the recombinant vector is introduced, and a method of producing the polypeptide using the recombinant microorganism.
생체 내에 존재하는 다양한 물질 중에서, 단백질은 생명 현상의 유지 및 기능을 수행하는 거대 분자로서 넓은 범위의 생물학적 역할을 담당하고 있다. 이런 역할을 수행하기 위해서는 우선 단백질-단백질 간 상호작용이 이루어져야 하며, 이는 모든 생명현상의 기초라고 할 수 있다. 만약 단백질 상호작용이 제대로 조절되어지지 않게되면 체내의 항상성은 망가지게 되어 결과적으로 다양한 질병을 일으키게 된다. 따라서 단백질 상호작용을 인위적으로 조절함으로써 질병을 치료하기 위한 다양한 방법이 연구 및 개발되어 왔으며 실제로 치료 목적에 사용가능한 다양한 의약품들이 성공적으로 개발되어 왔다.Of the various substances present in vivo, proteins are macromolecules that perform the maintenance and function of life phenomena and play a wide range of biological roles. In order to perform this role, protein-protein interactions must first be made, which is the basis of all life phenomena. If protein interactions are not properly regulated, homeostasis in the body will be destroyed, resulting in a variety of diseases. Accordingly, various methods for treating diseases by artificially controlling protein interactions have been studied and developed, and various medicines that can be actually used for therapeutic purposes have been successfully developed.
형광단백질은 생체 내에서 빛을 내 단백질들이 생체에서 어떻게 작용하는지 관찰할 수 있는 단백질을 말한다. 이 단백질을 통해 추적하고자 하는 단백질에다 형광단백질의 유전자를 붙여 세포에 주입하면 형광단백질을 따라 단백질의 움직임과 위치, 성장과정 등을 쉽게 확인할 수 있다. 형광단백질은 화학적 염색을 하지 않고도 세포 내에서 또는 세포를 고정한 후에도 안정된 형광을 나타내기 때문에 다양한 연구에 적용될 수 있다. 형광단백질을 통해 이전에는 눈으로 관찰할 수 없었던 생체 내에서 일어나는 현상을 살펴볼 수 있게 되어 신경세포의 번성과정 또는 암세포의 확산과정, 그리고 알츠하이머병 환자의 뇌 신경세포의 파괴과정을 추적하는 등 세포생물학ㆍ분자생물학 연구자들에게 널리 사용되고 있다.Fluorescent proteins are proteins that can see how proteins in the body work in the light. If you add the fluorescent protein gene to the protein you want to trace through this protein, you can easily identify the movement, position and growth process of the protein along with the fluorescent protein. Fluorescent proteins can be applied to a variety of studies because they exhibit stable fluorescence even after intracellular or immobilization of cells without chemical staining. Fluorescent proteins allow us to observe in vivo phenomena that were previously unobserved by the eye. They are used to track the process of proliferation of neurons, the process of spreading cancer cells, and the destruction of brain nerve cells in patients with Alzheimer's disease. It is widely used by molecular biology researchers.
이렇게 형광단백질의 사용량이 증가하면서 형광단백질에 결합하는 항체의 중요도 역시 높아지고 있다. 그 중 적색 형광단백질은 그 방출 파장대가 길어 녹색 형광단백질보다 다양한 조직에 적용 가능하고 빛에 대한 안정성이 강하다는 장점이 있다. 기존의 적색 형광단백질과 결합하는 항체를 만들어 내기 위하여 현재 주로 사용되고 있는 기술은, 토끼나 생쥐와 같은 척추동물의 몸 속에 형광단백질(항원)을 주입하여 면역체계에 있는 B세포에 의해 항체를 만드는 방법 등이 있다. 하지만, 상기와 같은 방법은 항체 단백질을 생산할 동물 세포주를 구축해야 하는 번거로움이 있다는 것과 다른 단백질 생산 개체(대장균, 효모)들에 비해 생산성이 많이 낮다는 단점이 있다. As the amount of the fluorescent protein used increases, the importance of the antibody binding to the fluorescent protein is also increasing. Among them, the red fluorescent protein has a longer emission wavelength range, which is applicable to various tissues and has a strong stability against light than the green fluorescent protein. Techniques currently being used to produce antibodies that bind to existing red fluorescent proteins include injecting fluorescent proteins (antigens) into the body of vertebrate animals such as rabbits and mice to create antibodies by immune system B cells . However, such a method has a disadvantage that it is troublesome to construct an animal cell line for producing an antibody protein and that productivity is low compared with other protein producing entities (E. coli, yeast).
한편, 본 발명자들은 선행특허(KR2012-0019927)에서 기존의 항체를 대체할 수 있는 비 항체 단백질 골격(non-antibody protein scaffold)인 리피바디(repebody)를 성공적으로 개발하였다. 상기 리피바디는 LRR(Leucine-rich repeat) 구조를 갖는 인터날린의 N-말단과 상기 VLR(Variable Lymphocyte Receptor)의 구조의 유사성을 바탕으로 융합하여 컨세서스 디자인으로 최적화시킨 폴리펩타이드를 의미한다. 상기 리피바디는 크기가 항체의 1/5 수준이고, 대장균에서 대량생산 되며 동물 실험결과 면역원성이 거의 없다. 또한, 열 및 pH 안정성이 매우 우수하고, 타겟에 대한 결합력을 pico-mole 수준까지 매우 용이하게 증대시킬 수 있으며, 타겟에 대한 특이성이 매우 탁월하다.On the other hand, the present inventors have successfully developed a non-antibody protein scaffold repebody that can replace existing antibodies in the prior art (KR2012-0019927). The lipid body refers to a polypeptide optimized by fusion of the N-terminal of interleukin having an LRR (Leucine-rich repeat) structure and the VLR (Variable Lymphocyte Receptor) based on the similarity of structure to the consecutive design. The lipid body is 1/5 of the size of the antibody, and is produced in a large amount in E. coli. In addition, it has excellent heat and pH stability, can very easily increase the binding force to the target to the pico-mole level, and has excellent specificity to the target.
이에 본 발명자들은, 리피바디 골격을 이용하여 다양한 단백질에 결합력을 갖는 범용적인 결합 단백질을 개발하고자 예의 노력한 결과, 리피바디의 구조적 특징인 모듈성과 전체구조분석을 통해 구축한 무작위한 돌연변이 라이브러리를 바탕으로, 적색 형광단백질에 대해 결합력을 갖는 신규한 폴리펩타이드를 선별하고, 상기 폴리펩타이드가 적색 형광단백질에 선택적으로 결합 가능함을 확인하고, 본 발명을 완성하게 되었다.
Accordingly, the present inventors have made intensive efforts to develop a universal binding protein having binding ability to various proteins using the lipid body framework. As a result, based on the random mutant library constructed through analysis of the structural and structural characteristics of the lipid body, , A novel polypeptide having a binding ability to a red fluorescent protein was selected and it was confirmed that the polypeptide could selectively bind to a red fluorescent protein, thereby completing the present invention.
본 발명의 목적은 적색 형광단백질에 선택적으로 결합할 수 있는 신규 폴리펩타이드; 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드; 상기 폴리뉴클레오타이드를 포함하는 재조합 벡터; 상기 재조합 벡터가 도입되어 있는 재조합 미생물 및 상기 재조합 미생물을 이용하여 상기 폴리펩타이드를 생산하는 방법을 제공하는데 있다.
It is an object of the present invention to provide a novel polypeptide capable of selectively binding to a red fluorescent protein; A polynucleotide encoding said polypeptide; A recombinant vector comprising the polynucleotide; A recombinant microorganism into which the recombinant vector is introduced and a method of producing the polypeptide using the recombinant microorganism.
상기 목적을 달성하기 위하여, 본 발명은 알파 나선형 캡핑 모티프(capping mofit)를 가지는 LRR 패밀리 단백질의 N-말단과 VLR 단백질의 변형된 반복모듈 및 VLR 단백질의 C-말단이 융합되어 있는 적색 형광단백질에 선택적으로 결합하는 서열번호 1의 폴리펩타이드를 제공한다.In order to accomplish the above object, the present invention relates to a method for producing a red fluorescent protein having an alpha helical capping mofit, a modified repeating module of the VLR protein and an N-terminal of the LRR family protein, RTI ID = 0.0 > 1 < / RTI >
본 발명은 또한, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 재조합 벡터 및 상기 재조합 벡터가 도입된 재조합 미생물을 제공한다.The present invention also provides a polynucleotide encoding the polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant microorganism into which the recombinant vector is introduced.
본 발명은 또한, (a) 상기 재조합 미생물을 배양하여 배양물을 수득하는 단계; 및 (b) 상기 배양된 재조합 미생물 또는 배양물로부터 상기 폴리펩타이드를 회수하는 단계를 포함하는 적색 형광단백질에 선택적으로 결합하는 폴리펩타이드의 제조방법을 제공한다.
(A) culturing the recombinant microorganism to obtain a culture; And (b) recovering the polypeptide from the cultured recombinant microorganism or culture. The present invention also provides a method for producing a polypeptide that selectively binds to a red fluorescent protein.
본 발명의 폴리펩타이드는 적색 형광단백질에 대한 결합력이 우수하므로, 세포생물학, 분자생물학뿐만 아니라 기초 의과학 연구 분야에 효과적으로 활용될 수 있다.
Since the polypeptide of the present invention is excellent in binding ability to red fluorescent protein, it can be effectively used in basic biochemical research as well as cell biology and molecular biology.
도 1은 상기 선행특허(KR2012-0019927)에서 구축된 파지 라이브러리를 이용하여, 적색 형광단백질에 대한 파지 디스플레이의 바이오 패닝을 수행한 결과를 나타낸 것이다. BSA 대비 적색 형광단백질에 대한 효소면역측정법 (ELISA)에 의한 결합 신호를 평균화 (Normalization)를 하였으며, 이때 5배 이상의 신호가 증가한 클론에 대해 특이적인 결합력을 갖는 리피바디 클론으로 정의한다.
도 2는 Repebody의 전체 단백질 구조를 나타내는 개략도로서, 생체 분자를 인식하는 Concave와 구조 유지에 중요한 Convex지역으로 구분된다.
도 3은 패닝과정에서 선별된 Repebody B1의 기본 골격으로 변화된 아미노산 잔기의 위치를 그림으로 나타낸 것이다.
도 4는 선별된 repebody가 사람의 적색 형광단백질과 특이적으로 결합한다는 것을 보여주는 ELISA 결과를 나타낸 것이다.
도 5는 선별된 repebody와 적색 형광단백질의 결합력을 ITC로 측정한 결과를 나타낸 것이다.
도 6은 고체 표면에 흡착된 repebody-B1에 적색 형광단백질이 함유되어있는 세포배양액을 흘려준 뒤 생리학적 pH(pH7.4)에서 용출한 용액을 SDS-PAGE로 확인한 그림을 나타낸 것이다.
도 7은 HeLa 세포 내에서 repebody-B1이 EGFP(녹색 형광단백질)과 융합되어 수용성 단백질로 발현됨을 웨스턴 블럿 분석으로 확인한 그림을 나타낸 것이다.
도 8은 폴리펩타이드 B1과 mCherry(적색 형광단백질)이 동물 세포 내에서 결합함을 확인한 그림을 나타낸 것이다.
도 9는 폴리펩타이드 B1과 mOrange(적색 형광단백질)이 동물 세포 내에서 결합함을 확인한 그림을 나타낸 것이다. FIG. 1 shows the result of performing bio-panning of a phage display on a red fluorescent protein using a phage library constructed in the above-mentioned prior art (KR2012-0019927). The binding signal was normalized by enzyme immunoassay (ELISA) for red fluorescent protein relative to BSA, and defined as a lipid body clone having a specific binding specificity for a clone in which the signal increased 5 times or more.
Fig. 2 is a schematic diagram showing the whole protein structure of the repebody, which is divided into Concave that recognizes biomolecules and Convex which is important in structure maintenance.
FIG. 3 is a graph showing positions of amino acid residues changed into the basic skeleton of Repebody B1 selected in the panning process.
Figure 4 shows ELISA results showing that the selected repebody binds specifically to human red fluorescent protein.
FIG. 5 shows the results of ITC measurement of the binding strength between the selected repebody and the red fluorescent protein.
FIG. 6 is a graph showing SDS-PAGE of a solution eluted at physiological pH (pH 7.4) after flowing a cell culture solution containing red fluorescent protein in the repebody-B1 adsorbed on a solid surface.
FIG. 7 shows Western blot analysis showing that repebody-B1 is fused with EGFP (green fluorescent protein) in HeLa cells and expressed as a water-soluble protein.
Fig. 8 is a figure showing that the polypeptide B1 and mCherry (red fluorescent protein) are bound in animal cells.
Fig. 9 shows a figure confirming that polypeptide B1 and mOrange (red fluorescence protein) are bound in animal cells.
본 발명에서는 적색 형광단백질과 선택적으로 결합할 수 있는 신규 폴리펩타이드를 개발하는데 있어, 결합 특이성이 높고, 대량생산이 가능한 폴리펩타이드를 발굴하였다. 신규 폴리펩타이드를 코딩하는 폴리뉴클레오티드, 상기 폴기뉴클레오티드를 포함하는 재조합 벡터 및 상기 재조합 벡터가 도입된 재조합 미생물을 이용하여 적색 형광단백질과 결합하는 신규 폴리펩타이드를 확인하였다.In the present invention, in developing a novel polypeptide capable of selectively binding to a red fluorescent protein, a polypeptide having high binding specificity and capable of mass production has been discovered. A novel polypeptide that binds to a red fluorescent protein was identified using a polynucleotide encoding a novel polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant microorganism into which the recombinant vector was introduced.
따라서, 본 발명은 일 관점에서 알파 나선형 캡핑 모티프(capping mofit)를 가지는 LRR 패밀리 단백질의 N-말단과 VLR단백질의 변형된 반복모듈 및 VLR 단백질의 C-말단이 융합되어 있는 적색 형광단백질에 선택적으로 결합하는 서열번호 1의 폴리펩타이드에 관한 것이다.Accordingly, the present invention provides, in one aspect, a method for selectively modulating the N-terminus of an LRR family protein having an alpha helical capping mofit with a modified repetitive module of a VLR protein and a red fluorescent protein to which the C- Lt; RTI ID = 0.0 > 1 < / RTI >
본 발명의 일 실시예에서는 서열번호 1의 아미노산 서열을 포함하고, 적색 형광단백질과 효과적으로 결합할 수 있는 폴리펩타이드를 선별하였다.In one embodiment of the present invention, a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 and capable of effectively binding to a red fluorescent protein was selected.
본 발명자들은, 적색 형광단백질과 결합력이 높은 폴리펩타이드를 선별하기 위하여, 선행특허(KR2012-0019927)에서 구축된 파지 라이브러리를 이용하여, 바이오 패닝 기법으로 스크리닝 하였다. 상기 파지에서 적색 형광단백질과 특이적으로 결합하는 폴리펩타이드를 선별하고, 상기 폴리펩타이드의 아미노산 서열을 확인한 결과, 서열번호 3의 아미노산 서열에서 91번, 93번, 94번 187번, 189번 및 190번으로 이루어진 군으로부터 선택되는 하나 또는 둘 이상의 위치의 아미노산이 변이된 것을 특징으로 하는 적색 형광단백질에 선택적으로 결합하는 폴리펩타이드임을 확인하였다.The present inventors screened by using a biopanning technique using a phage library constructed in the prior art (KR2012-0019927) in order to select polypeptides having high binding ability with red fluorescent protein. The polypeptides specifically binding to the red fluorescent protein in the phage were selected and the amino acid sequences of the polypeptides were confirmed. As a result, amino acid sequences 91, 93, 94, 187, 189 and 190 in the amino acid sequence of SEQ ID NO: Wherein the amino acid at one or more positions selected from the group consisting of SEQ ID NOs: 1 to 3 is mutated.
본 발명의 용어 "적색 형광단백질(mCherry)"는 discosoma라는 산호로부터 추출된 적색형광 단백질의 한 종류로서 587 nm의 파장에 여기되어 610 nm의 형광을 방출한다. 분자량은 약 27,000인 단백질이고, 분자 모형은 11개의 베타 병풍 (beta sheet) 구조가 베타 베럴 (beta berrel) 을 이루며 형광 단백질의 위, 아래에는 각각 루프 구조(loop structure)가 존재한다. The term "red fluorescent protein (mCherry) " of the present invention is a kind of red fluorescent protein extracted from coral of discosoma and excites at a wavelength of 587 nm to emit fluorescence of 610 nm. Molecular weight is about 27,000. In the molecular model, eleven beta sheet structures form beta berrel, and loop structures exist above and below the fluorescent protein, respectively.
본 발명자들은 적색 형광단백질과 특이적으로 결합할 수 있는 신규한 폴리펩타이드를 개발하기 위하여, 인터날린 B (Internalin B) 단백질의 Ｎ-말단, 가변 림프구 수용체(Variable Lymphocyte Receptor, VLR) 의 LRR (Leucine-rich repeat) 단백질 부분이 융합된, 폴리펩타이드의 반복 모듈을 무작위적으로 포함하는 라이브러리를 구축하였다. 상기 라이브러리에 포함된 폴리펩타이드는 본 발명자의 선행특허(KR2012-0019927)에 기술된 서열번호 1의 폴리뉴클레오티드 서열에 의하여 코딩되거나 또는 해당 폴리뉴클레오티드 서열과 75%, 바람직하게는 85%, 보다 바람직하게는 90%, 더욱 바람직하게는 95% 이상의 상동성을 갖는 폴리뉴클레오티드 서열에 의하여 코딩될 수 있다. 또한, 상기 라이브러리는 상기 폴리뉴클레오티드를 포함하는 파지미드의 형태가 될 수 있다. The inventors of the present invention have conducted extensive studies to develop a novel polypeptide capable of specifically binding to a red fluorescent protein, in which the N-terminal of Internalin B protein, the LRR of a Variable Lymphocyte Receptor (VLR) -rich repeat) protein fragments were fused to construct a library randomly containing repetitive modules of the polypeptide. The polypeptide contained in the library may be coded according to the polynucleotide sequence of SEQ ID NO: 1 described in the prior art (KR2012-0019927) of the present inventor, or a polynucleotide sequence having 75%, preferably 85% May be encoded by a polynucleotide sequence having 90%, more preferably 95% or more homology. The library may also be in the form of a phagemid comprising the polynucleotide.
본 발명의 용어 "파지미드"란, 대장균을 숙주로 하는 바이러스인 파지로부터 유래된 원형의 폴리뉴클레오티드 분자를 의미하는데, 번식과 증식에 필요한 단백질 및 표면 단백질들의 서열이 포함되어 있다. 재조합 파지미드는 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 수행될 수 있다. 상기 파지미드는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 인핸서 같은 발현 조절 요소 외에도 분비를 위한 신호 서열 또는 리더 서열을 포함할 수 있고, 주로 원하는 단백질을 파지의 표면 단백질과 융합하여 파지 표면에 표지하기 위한 방법에 사용될 수 있다. 파지미드의 프로모터는 주로 유도성이며, 숙주세포를 선택하기 위한 선택성 마커를 포함할 수도 있다. 본 발명의 목적상 상기 파지미드는 상기 라이브러리를 구성하는 폴리펩타이드를 코딩하는 폴리뉴클레오티드르를 발현 및 분비하기 위한 신호 서열 또는 리더 서열인 MalEss, DsbAss 또는 PelBss를 포함하고, 파지의 표면에 재조합 단백질의 발현을 확인하기 위한 히스티딘-태그와 파지 표면으로의 발현을 위한 M13 파지의 표면 단백질의 일종인 gp3 도메인을 인코딩하는 폴리뉴클레오티드를 포함하는 상기 선행특허(KR2012-0019927)의 서열번호 2의 폴리뉴클레오티드가 될 수 있으나, 특별히 이에 제한되지는 않는다.The term "phagemid " of the present invention refers to a circular polynucleotide molecule derived from a phage, which is a host virus of Escherichia coli, and contains sequences of proteins and surface proteins necessary for reproduction and proliferation. The recombinant phagemid can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be carried out using enzymes generally known in the art or the like. The phagemid may contain a signal sequence or a leader sequence for secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, and an enhancer, and is preferably fused with the surface protein of the phage to be labeled on the phage surface Can be used for the method. The promoter of phagemid is mainly inducible and may contain a selectable marker to select the host cell. For the purpose of the present invention, the above-mentioned phagiimide includes MalEss, DsbAss or PelBss, which is a signal sequence or leader sequence for expressing and secreting a polynucleotide encoding a polypeptide constituting the library, and a recombinant protein The polynucleotide of SEQ ID NO: 2 of the foregoing patent (KR2012-0019927) comprising a histidine-tag for confirming expression and a polynucleotide encoding the gp3 domain, which is a kind of surface protein of M13 phage for expression on the phage surface, But is not limited thereto.
본 발명자들은 상기 파지미드를 포함하는 라이브러리를 이용한 파지 디스플레이 방법을 사용하여 적색 형광단백질에 대한 결합력이 우수한 신규한 폴리펩타이드(서열번호 1)를 선별하였다(도 3). The present inventors selected a novel polypeptide (SEQ ID NO: 1) having excellent binding ability to a red fluorescent protein (FIG. 3) using a phage display method using the library containing the phagemid.
본 발명의 용어 "인터날린 B 단백질"이란, 리스테리아(Listeria) 균주에서 발현되는 LRR 패밀리 단백질의 일종을 의미하는데, 다른 소수성 코어가 전 분자에 걸쳐서 일정하게 분포되어 있는 다른 LRR 패밀리 단백질들과는 다른 유형의 N-말단 구조 때문에 미생물에서도 안정적으로 발현되는 것으로 알려져 있다. 이러한 인터날린 단백질의 N-말단은 반복 모듈의 접힘(Folding)에 가장 중요한 N-말단 부위가 미생물 유래일 뿐 아니라 그 형태가 알파 나선을 포함하는 더욱 안정적인 구조를 포함하므로, 미생물에서 LRR 패밀리 단백질들의 안정적인 발현을 위하여 효과적으로 사용될 수 있다. The term " internulin B protein "of the present invention means a kind of LRR family protein expressed in a Listeria strain, wherein the other hydrophobic core is different in type from other LRR family proteins distributed uniformly throughout the molecule Because of its N-terminal structure, it is known to be stably expressed in microorganisms. The N-terminus of this interleukin protein contains a more stable structure in which the N-terminal region, which is most important for folding of the repetitive module, is derived from the microorganism and the morphology thereof includes the alpha helices. Can be effectively used for stable expression.
본 발명의 용어, "인터날린 단백질의 N-말단"은 단백질의 수용성 발현 및 접힘에 있어서 필요한 인터날린 단백질의 N-말단을 의미하며 알파 나선형 캡핑 모티프 (capping motif) 및 인터날린 단백질의 반복 모듈을 의미한다. 인터날린 단백질의 N-말단은 단백질의 수용성 발현 및 접힘에 있어서 필요한 인터날린 단백질의 N-말단은 제한 없이 포함되나, 그 예로 알파 나선형 캡핑 모티프인 "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE" 및 반복 모듈을 포함할 수 있다. 바람직하게 반복 모듈 패턴은 "LxxLxxLxLxxN"을 포함할 수 있다. 상기 반복 모듈 패턴 중 L은 알라닌, 글리신, 페닐알라닌, 티로신, 루이신, 이소루이신, 발린 또는 트립토판; N은 아스파라진, 글루타민, 세린, 시스테인 또는 트레오닌, x는 친수성 아미노산을 의미한다. 인터날린 단백질의 N-말단은 융합될 수 있는 LRR 패밀리 단백질의 종류에 따라 높은 구조 유사도를 갖는 N-말단이 선택될 수 있으며 결합 에너지 등의 계산을 통해 가장 안정적인 아미노산을 선택하여 해당 모듈의 아미노산의 변이가 가능하다.The term "N-terminus of interleven protein " of the present invention means the N-terminus of the interleukin protein necessary for water-soluble expression and folding of the protein and includes repeating modules of alpha helical capping motif and interlein protein it means. The N-terminus of the internulin protein includes, but is not limited to, the N-terminus of the interleukin protein necessary for the water-soluble expression and folding of the protein, for example the alpha helical capping motif "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE" and repetitive modules. Preferably, the repeating module pattern may include "LxxLxxLxLxxN ". Wherein L in the repeating module pattern is selected from the group consisting of alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan; N means asparagine, glutamine, serine, cysteine or threonine, and x means a hydrophilic amino acid. Depending on the type of LRR family protein that can be fused, the N-terminal of the internulin protein can be selected at the N-terminal with high structural similarity. The most stable amino acid can be selected by calculation of the binding energy and the like, Variation is possible.
본 발명의 용어 "가변 림프구 수용체 (Variable Lymphocyte Receptor, VLR)"란, 먹장어와 칠성 장어에서 발현되는 LRR 패밀리 단백질의 일종을 의미하는데, 먹장어와 칠성 장어에서 면역 기능을 수행하는 단백질로서 다양한 항원 물질에 결합할 수 있는 골격으로 유용하게 사용될 수 있다. 상기 인터날린 B 단백질의 N-말단 및 VLR 단백질이 융합된 폴리펩타이드는 인터날린 B 단백질이 융합되지 않은 VLR 단백질보다 수용성 및 발현양이 증가하므로, 이를 기반으로 한 신규한 단백질 치료제의 제조에 사용될 수 있다.The term " Variable lymphocyte receptor (VLR) "of the present invention refers to a type of LRR family protein expressed in Euphorbia and Echinochloa, It can be used as a skeleton that can be combined. The polypeptides in which the N-terminal and VLR proteins of the interleukin B protein are fused are more soluble and expressed in higher amounts than the VLR proteins in which the interlein B protein is not fused, so that they can be used in the manufacture of novel therapeutic agents have.
본 발명의 용어 "LRR 단백질"이란, 루이신이 일정한 위치에 반복되는 모듈의 조합으로 이루어진 단백질을 의미하는 것으로, (i) 하나 이상의 LRR 반복 모듈을 갖고 있고, (ii) 상기 LRR 반복 모듈은 20 내지 30개의 아미노산으로 이루어져 있고, (iii) LRR 반복 모듈은 보존 패턴으로 "LxxLxxLxLxxN"을 갖고 있으며, 여기서 L은 알라닌, 글리신, 페닐알라닌, 티로신, 루이신, 이소루이신, 발린 및 트립토판과 같은 소수성 아미노산을, N은 아스파라진, 글루타민, 세린, 시스테인 또는 쓰레오닌을, x는 임의의 의미하며, (iv) LRR 패밀리 단백질은 말발굽과 같은 삼차원 구조를 갖는 구조를 갖고 있는 단백질을 의미한다. 본 발명의 LRR 패밀리 단백질은 이미 그 서열이 알려져 있거나, 생체에서 새로 유도된 mRNA나 cDNA를 이용하여 찾아낸 것 뿐 아니라, 컨센서스 디자인 (Consensus design) 등의 설계를 통하여 자연계에 알려지지 않은 서열을 가지면서 반복모듈의 골격 구조가 있는 변이체를 모두 포함할 수 있다.The term "LRR protein" of the present invention means a protein consisting of a combination of modules in which loicin is repeated at a predetermined position, and (i) has one or more LRR repetition modules, (ii) (Iii) the LRR repetition module has conservative pattern "LxxLxxLxLxxN ", wherein L is a hydrophobic amino acid such as alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine and tryptophan , N is asparagine, glutamine, serine, cysteine, or threonine, x is arbitrary, and (iv) LRR family protein means a protein having a three-dimensional structure such as horseshoe. The LRR family protein of the present invention is not only found in a known sequence or newly derived mRNA or cDNA in a living body, but also has a sequence unknown to the natural world through design such as consensus design All of the mutants having the skeletal structure of the module can be included.
본 발명의 용어 "리피바디"란, LRR 구조를 갖는 상기 인터날린의 N-말단과 상기 VLR의 구조의 유사성을 바탕으로 융합하여 컨센서스 디자인으로 최적화시킨 폴리 폴리펩타이드이다. 리피바디 단백질은 구조상 오목한 지역 (Concave) 와 볼록한 지역 (Convex) 으로 나누어 질 수 있다(도 4). 여기서 오목한 지역은 서열의 다양성이 높으며 단백질 상호작용에 중요한 부위로 알려져 있다. 이와 반대로 볼록한 지역은 보존성이 높은 서열을 바탕으로 단백질의 전체구조를 안정하게 유지하는 역할을 수행한다. 리피바디 단백질은 반복 모듈을 갖는 LRR 패밀리에 속하는 모든 단백질을 상기 방법으로 수용성 발현 및 단백질의 생물리화학적 성질을 향상 시킨 모든 융합 LRR 패밀리 단백질을 모두 포함할 수 있다.The term "lipid body " of the present invention is a polypolypeptide that is fused to the consensus design based on the similarity of the structure of the VLR with the N-terminal of the interlein having the LRR structure. Lipid body proteins can be divided into structurally concave regions (Convex) and convex regions (Fig. 4). Here, concave regions are high in sequence diversity and are known to be important for protein interaction. Conversely, the convex region plays a role in maintaining the overall structure of the protein stably based on the highly conserved sequence. Lipid body proteins can include all of the fusion proteins of the LRR family with repetitive modules that are both water soluble in this way and all of the fused LRR family proteins that have improved the biochemical properties of the protein.
본 발명은 다른 관점에서, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 재조합 벡터 및 상기 재조합 벡터가 도입된 재조합 미생물에 관한 것이다.In another aspect, the present invention relates to a polynucleotide encoding the polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant microorganism into which the recombinant vector is introduced.
본 발명에서 제공하는 상기 폴리뉴클레오티드는 특별히 이에 제한되지 않으나, 상기 서열번호 1의 아미노산 서열을 코딩하는 서열번호 2의 폴리뉴클레오티드가 될 수 있고, 상기 폴리뉴클레오티드와 70% 이상, 보다 바람직하게는 80% 이상, 보다 바람직하게는 90% 이상의 상동성을 가지는 염기서열을 가지는 폴리뉴클레오티드일 수 있으나, 특별히 이에 제한되지는 않는다.The polynucleotide provided in the present invention may be a polynucleotide of SEQ ID NO: 2 which codes for the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and may be a polynucleotide of 70% or more, more preferably 80% Or more, more preferably 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 2, but is not particularly limited thereto.
본 발명의 용어 "벡터"는 적합한 숙주 내에서 목적 단백질을 발현시킬 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 상기 목적 단백질을 암호화하는 폴리뉴클레오티드의 염기서열을 함유하는 DNA 제조물을 의미한다. 상기 조절 서열은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함한다. 벡터는 적당한 숙주 내로 형질 전환된 후, 숙주 게놈과 무관하게 복제되거나 기능할 수 있으며, 게놈 그 자체에 통합될 수 있다.The term "vector" of the present invention means a DNA construct containing a nucleotide sequence of a polynucleotide encoding said target protein operably linked to a suitable regulatory sequence so as to be able to express the protein of interest in the appropriate host. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be transcribed into a suitable host, then replicated or functional, independent of the host genome, and integrated into the genome itself.
본 발명에서 사용되는 재조합 벡터는 숙주 중에서 복제 가능한 것이면 특별히 한정되지 않으며 당 업계에 알려진 임의의 벡터를 이용할 수 있다. 통상 사용되는 벡터의 예로는 천연 상태이거나 재조합된 상태의 플라스미드, 파지미드, 코스미드, 바이러스 및 박테리오파지를 들 수 있다. 예를 들어, 파지 벡터 또는 코스미드 벡터로서 pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, 및 Charon21A 등을 사용할 수 있으며, 플라스미드 벡터로서 pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등을 사용할 수 있다. 본 발명에서 사용 가능한 벡터는 특별히 제한되는 것이 아니며 공지된 발현 벡터를 사용할 수 있다. 바람직하게는 pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pET-21a, pET-32a 벡터 등을 사용할 수 있다. 가장 바람직하게는 pET-21a, pET-32a 벡터를 사용할 수 있다.The recombinant vector used in the present invention is not particularly limited as long as it is replicable in a host, and any vector known in the art can be used. Examples of commonly used vectors include plasmids in the natural or recombinant state, phagemids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR type, pUC type, pBluescriptII type , pGEM-based, pTZ-based, pCL-based, pET-based, and the like. The vector usable in the present invention is not particularly limited, and known expression vectors can be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pET-21a and pET-32a vectors can be used. Most preferably, pET-21a, pET-32a vectors can be used.
본 발명의 용어 "재조합 미생물"란, 하나 이상의 목적 단백질을 암호화하는 유전자를 갖는 재조합 벡터가 숙주세포에 도입되어 목적 단백질을 발현시키도록 형질이 감염된 세포를 의미하며, 진핵세포, 원핵세포 등의 모든 세포가 될 수 있는데, 특별히 이에 제한되지 않으나, 대장균, 스트렙토미세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있다. 본 발명에서 사용 가능한 숙주세포는 특별히 제한되는 것이 아니나, 바람직하게는 대장균을 숙주세포로 사용할 수 있다. 가장 바람직하게는 대장균 BL21(DE3), OrigamiB(DE3)를 숙주세포로 사용할 수 있다.The term "recombinant microorganism " in the present invention means a cell in which a recombinant vector having a gene encoding at least one target protein is introduced into a host cell to infect the target protein so as to express the target protein, and all of eukaryotic cells and prokaryotic cells Cells, including, but not limited to, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells. The host cell usable in the present invention is not particularly limited, but Escherichia coli can be preferably used as a host cell. Most preferably, Escherichia coli BL21 (DE3) and Origami B (DE3) can be used as host cells.
본 발명에서 용어 "재조합"이란, 표적 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 재조합 벡터를 숙주 세포 내에 도입하여 숙주세포 내에서 상기 폴리뉴클레오티드가 암호화하는 단백질이 발현할 수 있도록 하는 것을 의미한다. 형질 전환된 폴리뉴클레오티드는 숙주세포 내에 발현될 수 있기만 한다면, 숙주세포의 염색체 내에 삽입되어 위치하거나 염색체 외에 위치하든지 상관없이 이들 모두를 포함한다. 또한, 상기 폴리뉴클레오티드는 표적 단백질을 암호화하는 DNA 및 RNA를 포함한다. 상기 폴리뉴클레오티드는 숙주세포 내로 도입되어 발현될 수 있는 것이면, 어떠한 형태로 도입되는 것이든 상관없다. 예를 들면, 상기 폴리뉴클레오티드는, 자체적으로 발현되는데 필요한 모든 요소를 포함하는 유전자 구조체인 발현 카세트 (Expression cassette)의 형태로 숙주세포에 도입될 수 있다. 상기 발현 카세트는 통상 상기 폴리뉴클레오티드에 작동 가능하게 연결되어 있는 프로모터(promoter), 전사 종결 신호, 리보좀 결합부위 및 번역 종결신호를 포함한다. 상기 발현 카세트는 자체 복제가 가능한 재조합 벡터 형태일 수 있다. 또한, 상기 폴리뉴클레오티드는 그 자체의 형태로 숙주세포에 도입되어, 숙주세포에서 발현에 필요한 서열과 작동 가능하게 연결되어 있는 것일 수도 있다.The term "recombinant" in the present invention means introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell so that a protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotides include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, so long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as far as it is capable of being introduced into a host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. The expression cassette typically includes a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a recombinant vector that is self replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell.
본 발명은 또 다른 관점에서, (a) 상기 재조합 미생물을 배양하여 배양물을 수득하는 단계; 및 (b) 상기 배양된 재조합 미생물 또는 배양물로부터 상기 폴리펩타이드를 회수하는 단계를 포함하는 적색 형광단백질에 선택적으로 결합하는 폴리펩타이드 제조방법에 관한 것이다.In another aspect, the present invention provides a method for producing a recombinant microorganism, comprising: (a) culturing the recombinant microorganism to obtain a culture; And (b) recovering the polypeptide from the cultured recombinant microorganism or culture. The present invention also relates to a method for producing a polypeptide that selectively binds to a red fluorescent protein.
본 발명에 있어서, 상기 재조합 미생물을 배양하는 단계는 특별히 이에 제한되지 않으나, 공지된 회분식 배양방법, 연속식 배양방법, 유가식 배양방법 등에 의해 수행됨이 바람직하고, 배양조건은 특별히 이에 제한되지 않으나, 염기성 화합물(예: 수산화나트륨, 수산화칼륨 또는 암모니아) 또는 산성 화합물(예: 인산 또는 황산)을 사용하여 적정 pH(pH 5 내지 9, 바람직하게는 pH 6 내지 8, 가장 바람직하게는 pH 6.8)를 조절할 수 있고, 산소 또는 산소-함유 가스 혼합물을 배양물에 도입시켜 호기성 조건을 유지할 수 있으며, 배양온도는 20 내지 45℃, 바람직하게는 25 내지 40℃를 유지할 수 있고, 약 10 내지 160 시간동안 배양함이 바람직하다. 상기 배양에 의하여 생산된 상기 폴리펩타이드는 배지중으로 분비되거나 세포내에 잔류할 수 있다.In the present invention, the step of culturing the recombinant microorganism is not particularly limited, but is preferably carried out by a known batch culture method, a continuous culture method, a fed-batch culture method and the like, and the culture conditions are not particularly limited, (PH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8) using a basic compound such as sodium hydroxide, potassium hydroxide or ammonia or an acidic compound such as phosphoric acid or sulfuric acid. And the oxygen or oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions and the incubation temperature can be maintained at 20 to 45 캜, preferably 25 to 40 캜, for about 10 to 160 hours . The polypeptide produced by the culture may be secreted into the medium or left in the cells.
본 발명에서, 사용되는 배양용 배지는 탄소 공급원으로는 당 및 탄수화물(예: 글루코오스, 슈크로오스, 락토오스, 프럭토오스, 말토오스, 몰라세, 전분 및 셀룰로오스), 유지 및 지방(예: 대두유, 해바라기씨유, 땅콩유 및 코코넛유), 지방산(예: 팔미트산, 스테아르산 및 리놀레산), 알콜(예: 글리세롤 및 에탄올) 및 유기산(예: 아세트산) 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있고; 질소 공급원으로는 질소-함유 유기 화합물(예: 펩톤, 효모 추출액, 육즙, 맥아 추출액, 옥수수 침지액, 대두 박분 및 우레아), 또는 무기 화합물(예: 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄) 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있으며; 인 공급원으로서 인산 이수소칼륨, 인산수소이칼륨, 이에 상응하는 나트륨 함유 염 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있고; 기타 금속염(예: 황산마그네슘 또는 황산철), 아미노산 및 비타민과 같은 필수성장-촉진 물질을 포함할 수 있다.In the present invention, the culture medium to be used includes carbon sources such as sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, fats and oils such as soybean oil, Sunflower seed oil, peanut oil and coconut oil), fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, Can be; Examples of nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium nitrate) may be used individually or in combination; As the phosphorus source, potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salt may be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, amino acids and vitamins.
본 발명의 상기 배양 단계에서 생산된 폴리펩타이드를 회수하는 방법은 배양방법, 예를 들어 회분식, 연속식 또는 유가식 배양 방법 등에 따라 당해 분야에 공지된 적합한 방법을 이용하여 배양액으로부터 목적하는 폴리펩타이드를 수집할 수 있다.
The method for recovering the polypeptide produced in the culturing step of the present invention can be carried out by culturing the desired polypeptide from the culture solution using a suitable method known in the art according to a culture method, for example, a batch method, a continuous method or a fed- Can be collected.
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
실시예 1: 무작위적인 파지 라이브러리를 통한 적색 형광단백질과 특이적으로 결합하는 폴리펩타이드 선별 Example 1: Screening for polypeptides that specifically bind to red fluorescent protein via a random phage library
실시예 1-1: 리피바디 라이브러리의 패닝 과정을 통한 적색 형광단백질과 결합하는 폴리펩타이드 선별Example 1-1: Screening of a polypeptide binding to a red fluorescent protein through a panning process of a lipid body library
상기 선행특허(KR2012-0019927)에서 구축한 라이브러리를 사용하여 적색 형광단백질에 결합할 수 있는 폴리펩타이드를 선별하여 정제하였다. 적색 형광단백질과 결합할 수 있는 후보를 선별하기 위하여, 적색 형광단백질을 면역튜브 (Immuno-tube)에 100 ㎍/㎖의 농도로 가하고, 4 ℃에서 12시간동안 코팅하였다. 상기 코팅된 면역튜브를 PBS로 3회 세척하고, 1% BSA와 0.1 % Tween 20을 포함하는 PBS 용액 (TPBSA)으로 4 ℃에서 2시간동안 블로킹(Blocking) 하였다. 그런 다음, 상기 정제된 파지를 1012 cfu/ml의 농도로 상기 코팅된 면역튜브에 가하고, 상온에서 2시간 반응시켰다. 반응이 종료된 후, 0.1% Tween 20을 포함하는 PBS 용액 (TPBS)으로 총 2분간 5번씩 및 PBS로 2번 세척하였다. 마지막으로, 1 ml 0.2 M Gly-HCl (pH 2.2)를 상기 면역튜브에 가하고, 상온에서 10분간 반응시킴으로써, 적색 형광단백질과 결합할 수 있는 리피바디 후보를 표면에 발현시킨 파지를 용출시켰다. 상기 용출액에 60 ㎕의 1.0 M Tris-HCl (pH 9.1)를 가하여 중화시키고, 숙주세포인 10ml 대장균 XL1-Blue 용액 (OD600 = 0.5)에 가한 다음, 2xYT 플레이트에 도말하는 바이오패닝 (Bio-panning) 과정을 동일하게 4회 반복하여 수행하였다. 그 결과, 각 패닝 과정을 통해 적색 형광단백질과 특이적으로 결합하는 파지가 농축됨을 확인하였다. 상기 결과는 적색 형광단백질와 결합하는 라이브러리 파지가 특이적으로 증가함을 의미하는 것으로 분석되었다.
The library constructed in the foregoing patent (KR2012-0019927) was used to select and purify polypeptides capable of binding to the red fluorescent protein. To select candidates capable of binding to red fluorescent proteins, red fluorescent proteins were added to an immuno-tube at a concentration of 100 μg / ml and coated at 4 ° C for 12 hours. The coated immunotubes were washed three times with PBS and blocked with PBS solution (TPBSA) containing 1% BSA and 0.1
실시예 1-2: 선별된 리피바디의 적색 형광단백질에 대한 특이적인 결합여부 확인 및 서열 분석Example 1-2: Identification and Sequence Analysis of Specific Lipid Binding to Red Fluorescent Protein of Selected Lipid Bodies
실시예 1-1의 방법을 통하여 선별된 파지를 적색 형광단백질과 BSA가 코팅된 96-웰 플레이트를 이용하여 ELISA를 수행함으로써, BSA 대비 적색 형광단백질의 흡광도(OD450)가 25배 이상 높은 5개의 리피바디 후보를 선별하고 (도 1), 이들의 아미노산 서열을 확인하였다. 그 결과, 상기 선별된 파지에서 발현되는 적색 형광단백질과 특이적으로 결합하는 단백질의 아미노산 서열을 확인하였다. 즉, 91번 아미노산인 트레오닌이 아스파라진으로 치환되고, 93번 아미노산인 글루탐산이 글리신으로 치환되며, 94번 아미노산인 프롤린이 글루타민으로 치환되고, 187번 아미노산인 아르지닌이 프롤린으로 치환되며, 189번 아미노산인 티로신이 메타오닌으로 치환되고, 190번 아미노산인 글루타민이 라이신으로 치환되었음을 확인하였다(도 3). 상기 결과는 적색 형광단백질과 결합하는데 중요한 역할을 수행하는 잔기가 존재함을 의미하는 것으로 분석되었다.
ELISA was performed using a 96-well plate coated with red fluorescent protein and BSA on the phage selected by the method of Example 1-1 to determine the absorbance (OD450) of the red fluorescent protein relative to BSA was 5 Lipid body candidates were selected (Fig. 1), and their amino acid sequences were confirmed. As a result, the amino acid sequence of the protein specifically binding to the red fluorescent protein expressed in the selected phage was confirmed. That is, threonine, which is amino acid 91, is replaced with asparagine, glutamic acid which is 93 amino acid is replaced by glycine, proline which is 94 amino acid is replaced by glutamine, arginine which is amino acid 187 is substituted by proline, It was confirmed that the amino acid tyrosine was substituted with methionine and that the amino acid 190 was substituted with lysine (FIG. 3). The results were analyzed to indicate the presence of a residue that plays an important role in binding to the red fluorescent protein.
실시예 2: 선별된 Repebody의 적색 형광단백질 에 대한 특이적인 결합 특성 분석Example 2: Analysis of specific binding characteristics of selected red fluorescent protein
ELISA 결과를 바탕으로 Repebody B1을 대상으로 하여, 본 발명자들은 추가적으로 다양한 종들의 적색 형광단백질 과 BSA을 코팅한 플레이트를 이용하여 ELISA를 수행하여 상기의 Repebody B1이 적색 형광단백질 에 대한 표적 특이성을 갖고 있음을 알 수 있었다(도 1). Based on the results of the ELISA, the present inventors conducted an ELISA using a plate coated with red fluorescent protein and BSA in addition to various species, and found that the above-mentioned Repebody B1 has a target specificity for a red fluorescent protein (Fig. 1).
한편, 적색 형광단백질에 대한 상기 Repebody B1의 해리상수 (Dissociation constant)를 측정하였다. PBS에 0.2 mM(6 mg/ml)로 용해된 Repebody와 PBS에 0.02 mM(3 mg /ml)로 용해된 적색 형광단백질을 사용하고, 등온열량측정기(Isothermal titration calorimetry, ITC)를 이용하여, 상온에서 적색 형광단백질 에 대한 상기 repebody B1의 해리상수를 측정하였다(도 5). 적색 형광단백질 에 대한 Repebody B1의 해리상수 (KD)는 34 nM임을 확인하였다.
Meanwhile, the dissociation constant of the above-mentioned Repebody B1 against the red fluorescent protein was measured. The red fluorescent protein dissolved in PBS at 0.2 mM (6 mg / ml) and 0.02 mM (3 mg / ml) in PBS was used and the isothermal titration calorimetry (ITC) The dissociation constant of the repebody B1 for the red fluorescent protein was measured (Fig. 5). The dissociation constant (K D ) of the red fluorescent protein was determined to be 34 nM for Repebody B1.
실시예 3: 세포 내에서 선별된 Repebody-B1의 적색 형광단백질에 대한 특이적인 결합 특성 분석Example 3: Analysis of specific binding characteristics of red fluorescent protein in selected cells of Repebody-B1
실시예 3-1: 동물 세포 내에서 Repebody 단백질의 발현을 위한 재조합 벡터 제작 Example 3-1: Production of recombinant vector for expression of Repebody protein in animal cells
실시예 2에서 최종 결정된 Repebody B1의 폴리뉴클레오티드를 pEGFP-N1(Clontech, USA) 벡터에 삽입하여 Repebody-EGFP로 명명되는 컨스트럭트를 제작하였다.
The polynucleotide of Repebody B1 finally determined in Example 2 was inserted into pEGFP-N1 (Clontech, USA) vector to construct a construct named Repebody-EGFP.
실시예 3-2 동물 세포 내에서 Repebody-EGFP 단백질의 발현을 확인Example 3-2 Expression of Repebody-EGFP Protein in Animal Cells
실시예 3-1에서 제조한 Repebody-EGFP 로 HeLa세포를 형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37?, 5% CO2 조건에서 24시간 배양한 HeLa 세포를 추출하였다. 세포 분쇄액 내의 단백질을 12% SDS 폴리아크릴아마이드 젤로 분리한 다음 젤에 있는 단백질을 나이트로셀룰로스 막으로 전기이동시키고 단백질이 이동된 나이트로셀룰로스 막을 0.5% 소혈청알부민이 들어있는 TBST 완충액으로 1시간 실온에서 블로킹시킨 후, 막을 1차 항체로 1시간 반응시켰다. 1차 항체를 TBST 완충액으로 세척한 다음 퍼옥시데이즈가 결합된 항염소 IgG 항체(Biorad, 1:3,000 dilution)로 1 시간 반응시키고 ECL 키트를 이용하여 단백질에 반응하는 단백질 띠를 확인하였다.(도 7)HeLa cells were transformed with the Repebody-EGFP prepared in Example 3-1, and then HeLa cells were cultured in DMEM medium containing 500 μg / ml of G418 for 24 hours at 37 ° C and 5% CO 2 . The protein in the cell lysate was separated by 12% SDS polyacrylamide gel, the protein in the gel was electrophoretically transferred to nitrocellulose membrane, and the nitrocellulose membrane in which the protein was transferred was washed with TBST buffer containing 0.5% bovine serum albumin for 1 hour After blocking at room temperature, the membrane was reacted with primary antibody for 1 hour. The primary antibody was washed with TBST buffer, reacted with peroxidase-conjugated anti-chlorine IgG antibody (Biorad, 1: 3,000 dilution) for 1 hour, and protein bands reacted with the protein were identified using an ECL kit 7)
실시예 3-3: EB(End Binding Protein)3- mCherry-N1 컨스트럭트의 제작Example 3-3: Fabrication of EB (End Binding Protein) 3-mCherry-N1 construct
mCherry 형광단백질이 포함된 pmCherry-N1(Clontech, USA) 벡터에서 mCherry 앞에 EB3을 융합하여 EB3-mCherry-N1 컨스트럭트를 제작하였다.
The EB3-mCherry-N1 construct was constructed by fusing EB3 in front of mCherry in the pmCherry-N1 (Clontech, USA) vector containing the mCherry fluorescent protein.
실시예 3-4: EB(End Binding Protein )1- mOrange-N1 컨스트럭트의 제작Example 3-4: Fabrication of EB (End Binding Protein) 1-mOrange-N1 construct
mOrange 형광단백질이 포함된 pmOrange-N1(Clontech, USA) 벡터에서 mCherry 앞에 EB3을 융합하여 EB1-mOrange-N1 컨스트럭트를 제작하였다.
EB1-mOrange-N1 construct was constructed by fusing EB3 in front of mCherry in pmOrange-N1 (Clontech, USA) vector containing mOrange fluorescent protein.
실시예 3-5: 세포 내에서 Repebody의 적색 형광단백질에 대한 특이적인 결합 특성 분석Example 3-5: Specific binding characteristics of the red fluorescent protein of the repebody in the cells
실시예 3-1 및 3-3에서 제조한 Repebody-EGFP와 EB3-mCherry-N1 로 HeLa세포를 공형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 5% CO2 조건에서 24시간 배양한 배양한 뒤에 8웰 챔버 슬라이드의 배지챔버(media chamber)를 제거한 뒤 슬라이드를 PBS(pH 7.4)로 1분간 세척 후 4% 파라포름알데히드 용액 (paraformaldehyde solution)으로 10분간 고정하였다. 파장 488 nm의 빛의 조사 전후로 공초점현미경 이미지를 수득하였다(도 8). 그 결과, Repebody-EGFP 와 EB3-mCherry-N1 이 같은 위치에 존재하는 것을 확인하였다. 이러한 결과는 Repbody와 mCherry가 HeLa 세포 내에서 결합함을 의미한다. HeLa cells were co-transformed with Repebody-EGFP and EB3-mCherry-N1 prepared in Examples 3-1 and 3-3, and then cultured in DMEM medium containing 500 μg / ml of G418 at 37 ° C in 5% CO 2 For 24 hours, the media chamber of the 8 well chamber slides was removed and the slides were washed with PBS (pH 7.4) for 1 minute and fixed with 4% paraformaldehyde solution for 10 minutes. Confocal microscope images were obtained before and after irradiation of light with a wavelength of 488 nm (FIG. 8). As a result, it was confirmed that Repebody-EGFP and EB3-mCherry-N1 exist in the same position. These results indicate that Repbody and mCherry bind within HeLa cells.
이어, 실시예 3-1 및 3-4에서 제조한 Repebody-EGFP와 EB1-mOrange-N1 로 HeLa세포를 공형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 5% CO2 조건에서 24시간 배양한 배양한 뒤에 8웰 챔버 슬라이드의 배지챔버(media chamber)를 제거한 뒤 슬라이드를 PBS(pH 7.4)로 1분간 세척 후 4% 파라포름알데히드 용액 (paraformaldehyde solution)으로 10분간 고정하였다. 파장 488 nm의 빛의 조사 전후로 공초점현미경 이미지를 수득하였다(도 9). 그 결과, Repebody-EGFP 와 EB1-mOrange-N1 이 같은 위치에 존재하는 것을 확인하였다. 이러한 결과는 Repbody와 mOrange가 HeLa 세포 내에서 결합함을 의미한다.
Next, HeLa cells were cotransformed with Repebody-EGFP and EB1-mOrange-N1 prepared in Examples 3-1 and 3-4, and then cultured in DMEM medium containing 500 μg / ml of G418 at 37 ° C. in 5% CO 2 After incubation for 24 h, the media chamber of the 8 well chamber slides was removed. The slides were washed with PBS (pH 7.4) for 1 min and fixed with 4% paraformaldehyde solution for 10 min. Respectively. Confocal microscope images were obtained before and after irradiation of light with a wavelength of 488 nm (FIG. 9). As a result, it was confirmed that Repebody-EGFP and EB1-mOrange-N1 exist at the same position. These results indicate that Repbody and mOrange bind within HeLa cells.
이러한 결과를 통해 본 발명자들은, 세포 내에서 soluble하게 발현되고, mCherry 및 mOrange와 선택적으로 결합하는 신규 폴리펩타이드인 Repebody-B1을 발굴하였다. 이는 상기 Repebody가 mCherry 및 mOrange와 같은 적색 형광단백질의 표지자로 쓰일 수 있고 나아가 mCherry와 mOrange를 이용한 세포 소기관을 연구하는 실험 등에 유용하게 적용될 수 있음을 의미한다.
From these results, we have found a new polypeptide, soluble in cells, that selectively binds mCherry and mOrange, Repebody-B1. This means that the above-mentioned Repebody can be used as a marker of red fluorescence proteins such as mCherry and mOrange, and further can be applied to experiments for studying organelles using mCherry and mOrange.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> KAIST <120> Novel Polypeptides Binding with red fluorescent protein <130> P14-B222 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody-B1 <400> 1 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp 1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr 20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala 35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Lys Leu Thr Leu Val Ser Asn Lys Leu His Asp Ile Ser Ala 65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Asn Leu Gly Gln Asn Gln 85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Gln Leu Trp Ala Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala Phe Asn Gln 130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Met Lys Asn Gln 180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln 195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile 210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly 225 230 235 240 Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile 245 250 255 Ile Cys Pro Thr 260 <210> 2 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> Repebody-B1 <400> 2 gaaaccatta ccgtgagcac cccgatcaaa cagatttttc cggatgacgc gttcgccgaa 60 acgatcaaag caaacctgaa gaaaaagagc gttaccgatg ctgtcacgca aaatgaactg 120 aacagtattg accagatcat tgcgaataac tccgatatca aatcagtgca aggcattcag 180 tatctgccga atgttcgtaa gctgacgctg gtgagtaaca aactgcatga catctcggca 240 ctgaaagaac tgaccaatct gacgtatctg aatctggggc agaaccaact gcagtcactg 300 ccgaacggcg tgtttgataa actgacgaac ctgaaagaac tgcagctgtg ggcgaatcaa 360 ctgcagtctc tgccggatgg tgtgttcgac aaactgacca acctgacgta tctgaatctg 420 gcttttaacc aactgcagag tctgccgaaa ggtgtctttg acaaactgac caatctgacg 480 gaactggatc tgtcctataa ccaactgcag tcactgccga aaggtgtctt cgataaactg 540 acccagctga aagatctgag gctgatgaag aatcagctga aatcggtccc ggacggcgtg 600 tttgatcgtc tgaccagcct gcagtatatc tggctgcatg ataacccgtg ggattgcacc 660 tgtccgggta ttcgctacct gtctgaatgg atcaataaac acagtggcgt tgtcggcggc 720 ccggattcgg cgaaatgctc cggcagcggt aaaccggtgc gtagcattat ttgcccgacc 780 780 <210> 3 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody-B1-mutant <400> 3 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp 1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr 20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala 35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Lys Leu Thr Leu Val Ser Asn Lys Leu His Asp Ile Ser Ala 65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Thr Leu Glu Pro Asn Gln 85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Gln Leu Trp Ala Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala Phe Asn Gln 130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln 180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln 195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile 210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly 225 230 235 240 Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile 245 250 255 Ile Cys Pro Thr 260 <110> KAIST <120> Novel Polypeptide Binding with red fluorescent protein <130> P14-B222 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody-B1 <400> 1 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp 1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr 20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala 35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Lys Leu Thr Leu Val Ser Asn Lys Leu His Asp Ile Ser Ala 65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Asn Leu Gly Gln Asn Gln 85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Gln Leu Trp Ala Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala Phe Asn Gln 130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Met Lys Asn Gln 180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln 195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile 210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly 225 230 235 240 Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile 245 250 255 Ile Cys Pro Thr 260 <210> 2 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> Repebody-B1 <400> 2 gaaaccatta ccgtgagcac cccgatcaaa cagatttttc cggatgacgc gttcgccgaa 60 acgatcaaag caaacctgaa gaaaaagagc gttaccgatg ctgtcacgca aaatgaactg 120 aacagtattg accagatcat tgcgaataac tccgatatca aatcagtgca aggcattcag 180 tatctgccga atgttcgtaa gctgacgctg gtgagtaaca aactgcatga catctcggca 240 ctgaaagaac tgaccaatct gacgtatctg aatctggggc agaaccaact gcagtcactg 300 ccgaacggcg tgtttgataa actgacgaac ctgaaagaac tgcagctgtg ggcgaatcaa 360 ctgcagtctc tgccggatgg tgtgttcgac aaactgacca acctgacgta tctgaatctg 420 gcttttaacc aactgcagag tctgccgaaa ggtgtctttg acaaactgac caatctgacg 480 gaactggatc tgtcctataa ccaactgcag tcactgccga aaggtgtctt cgataaactg 540 acccagctga aagatctgag gctgatgaag aatcagctga aatcggtccc ggacggcgtg 600 tttgatcgtc tgaccagcct gcagtatatc tggctgcatg ataacccgtg ggattgcacc 660 tgtccgggta ttcgctacct gtctgaatgg atcaataaac acagtggcgt tgtcggcggc 720 ccggattcgg cgaaatgctc cggcagcggt aaaccggtgc gtagcattat ttgcccgacc 780 780 <210> 3 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody-B1-mutant <400> 3 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp 1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr 20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala 35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Lys Leu Thr Leu Val Ser Asn Lys Leu His Asp Ile Ser Ala 65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Thr Leu Glu Pro Asn Gln 85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Gln Leu Trp Ala Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala Phe Asn Gln 130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln 180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln 195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile 210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly 225 230 235 240 Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile 245 250 255 Ile Cys Pro Thr 260
Claims (11)
A polypeptide represented by the amino acid sequence of SEQ ID NO: 1 and selectively binding to a red fluorescent protein.
A polynucleotide encoding the polypeptide of claim 1.
A recombinant microorganism into which the polynucleotide of claim 7 has been introduced.
A recombinant microorganism into which the recombinant vector of claim 8 has been introduced.
(a) 제9항 또는 제10항의 재조합 미생물을 배양하여 제1항의 폴리펩타이드를 생성시키는 단계; 및
(b) 상기 생성된 폴리펩타이드를 회수하는 단계.A method for producing a polypeptide selectively binding to a red fluorescent protein comprising the steps of:
(a) culturing the recombinant microorganism of claim 9 or 10 to produce the polypeptide of claim 1; And
(b) recovering the resulting polypeptide.
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Non-Patent Citations (2)
| Title |
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| Binz H. K. et al., Nature Biotechnoloty 23(10): pp.1257-1268 (2005.10. 6.)* |
| Lee S-C., et al., Proc. Natl. Acad. Sci. 109(9):pp. 3299-3304 (2012. 2.28)* |
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