KR20240045048A - A tag for enhanced expression of membrane protein and use thereof - Google Patents
A tag for enhanced expression of membrane protein and use thereof Download PDFInfo
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- KR20240045048A KR20240045048A KR1020220177866A KR20220177866A KR20240045048A KR 20240045048 A KR20240045048 A KR 20240045048A KR 1020220177866 A KR1020220177866 A KR 1020220177866A KR 20220177866 A KR20220177866 A KR 20220177866A KR 20240045048 A KR20240045048 A KR 20240045048A
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Abstract
본 발명의 목적은 막 단백질의 발현을 증가시키기 위한 태그 및 이의 용도를 제공하는 것이다. 구체적으로, 본 발명에 따른 태그는 다양한 종류의 GPCR 단백질 발현을 무세포 시스템이나 대장균에서 유의적으로 증가시킴으로써, GPCR과 같은 막 단백질을 대장균에서 발현시키는데 유용하게 사용될 수 있다.An object of the present invention is to provide a tag and its use for increasing the expression of membrane proteins. Specifically, the tag according to the present invention can be usefully used to express membrane proteins such as GPCRs in E. coli by significantly increasing the expression of various types of GPCR proteins in cell-free systems or E. coli.
Description
본 발명은 막 단백질의 발현을 증가시키기 위한 태그 및 이의 용도에 관한 것이다.The present invention relates to tags and their use for increasing the expression of membrane proteins.
유전자 재조합 기술이 발달되면서 동물세포, 효모와 같은 진핵생물(eukaryote), 및 대장균과 같은 원핵생물(prokaryote) 등을 이용하여 유용한 목적 단백질을 생산하는 기술이 발전하고 있다. 또한, 이와 같이 생산된 재조합 단백질은 의약품 등을 포함하는 생물공학 산업에 널리 사용되고 있다.As genetic recombination technology develops, technology for producing useful target proteins using animal cells, eukaryotes such as yeast, and prokaryotes such as E. coli is advancing. In addition, recombinant proteins produced in this way are widely used in the biotechnology industry, including pharmaceuticals.
생화학적으로 모든 생물은 동일한 유전자 단위의 구성과 기능을 갖기 때문에 유전자를 재조합하여 이종 숙주에서 단백질을 생산하는 것이 가능하다. 유전자는 기능적으로 DNA에서 mRNA를 합성하는 전사(transcription) 과정과 mRNA에서 단백질을 합성하는 번역(translation) 과정을 각각 담당하는 부분으로 나눌 수 있다. 번역 과정에서 실제로 단백질로 발현되는 부분인 ORF(open reading frame)는 발현벡터에 삽입되어 기능적 유전자 단위를 완성함으로써 재조합 단백질의 생산을 가능하게 한다. 따라서, 발현벡터에서 단백질의 발현을 조절할 수 있는 요소들이 개발되어 목적 단백질의 특성에 맞춰 발현 시점과 발현양을 조절할 수 있다. 그러나, 유전자의 발현 효율은 항상 동일하지 않기 때문에 목적 단백질의 종류에 따라 최적 조건을 확립하기 위한 필요가 있다. 특히 목적 단백질이 안정성이나 접힘 등의 문제로 발현이 잘 되지 않거나, 낮은 가용성을 보이는 경우에 재조합 단백질을 생산하는데 어려움이 있다. 따라서, 효율적인 재조합 단백질의 생산은 단백질을 과발현시키면서 가용성(solubility)이 높은 형태를 갖도록 하는 것이다.Biochemically, all organisms have the same composition and function of genetic units, so it is possible to produce proteins in heterogeneous hosts by recombining genes. Genes can be functionally divided into parts responsible for the transcription process, which synthesizes mRNA from DNA, and the translation process, which synthesizes proteins from mRNA. ORF (open reading frame), which is the part that is actually expressed as a protein during the translation process, is inserted into the expression vector to complete the functional genetic unit, enabling the production of recombinant proteins. Therefore, elements that can control the expression of proteins in expression vectors have been developed, so that the expression time and amount can be adjusted according to the characteristics of the target protein. However, since the expression efficiency of genes is not always the same, there is a need to establish optimal conditions depending on the type of target protein. In particular, there are difficulties in producing recombinant proteins when the target protein is not expressed well due to problems such as stability or folding, or has low solubility. Therefore, efficient production of a recombinant protein involves overexpressing the protein so that it has a highly soluble form.
일반적으로 단백질의 발현 효율을 높이기 위해서는 벡터, 숙주, ORF 수준에서 조작이 시도되어 왔다. 그러나, 최근에는 전형적인 이들 요소 외에 새로운 조절인자들이 발굴되어 발현 효율의 증가를 위해 다양한 방법이 시도되고 있다. 그중 하나로서 단백질 접힘(folding) 또는 회수(recovery)를 용이하게 하는 기능성 태그(tag)가 장착된 벡터를 이용하여 목적 단백질의 가용성을 높이거나, 정제과정의 용이성을 제공하는 방법이 관심받고 있다.In general, manipulation has been attempted at the vector, host, and ORF levels to increase protein expression efficiency. However, recently, in addition to these typical factors, new regulatory factors have been discovered, and various methods are being attempted to increase expression efficiency. As one of them, a method of increasing the solubility of a target protein or providing ease of purification process by using a vector equipped with a functional tag that facilitates protein folding or recovery is attracting attention.
관련하여, 대한민국 등록특허 제10-2106773호는 목적 단백질의 수용성 및 발현량 증가를 위한 융합 태그 및 이의 용도에 관한 것으로, 특정 서열로 구성된 융합 태그가 포함된 발현벡터는 목적 단백질의 수용성 및 발현량이 증가됨을 개시하고 있다.In relation to this, Republic of Korea Patent No. 10-2106773 relates to a fusion tag for increasing the water solubility and expression level of a target protein and its use. An expression vector containing a fusion tag composed of a specific sequence increases the water solubility and expression level of the target protein. It is starting to increase.
본 발명의 목적은 막 단백질의 발현을 증가시키기 위한 태그 및 이의 용도를 제공하는 것이다.An object of the present invention is to provide a tag and its use for increasing the expression of membrane proteins.
상기 목적을 달성하기 위하여, 본 발명은 아데닌(adenine, A) 및 티민(thymine, T)으로 구성되고, 3 내지 99개의 뉴클레오타이드로 구성되는 막 단백질(membrane protein) 발현 증진용 태그(tag)를 제공한다.In order to achieve the above object, the present invention provides a tag for enhancing the expression of a membrane protein composed of adenine (A) and thymine (T) and composed of 3 to 99 nucleotides. do.
또한, 본 발명은 상기 막 단백질 발현 증진용 태그가 포함된 발현벡터를 제공한다.Additionally, the present invention provides an expression vector containing a tag for enhancing expression of the membrane protein.
또한, 본 발명은 상기 발현벡터가 형질전환된 숙주세포를 제공한다.Additionally, the present invention provides host cells transformed with the expression vector.
나아가, 본 발명은 상기 숙주세포에서 막 단백질을 발현시키는 단계를 포함하는 막 단백질의 발현을 증가시키는 방법을 제공한다.Furthermore, the present invention provides a method for increasing the expression of a membrane protein comprising expressing the membrane protein in the host cell.
본 발명에 따른 태그는 다양한 종류의 GPCR 단백질 발현을 무세포 시스템이나 대장균에서 유의적으로 증가시킴으로써, GPCR과 같은 막 단백질을 대장균에서 발현시키는데 유용하게 사용될 수 있다.The tag according to the present invention can be usefully used to express membrane proteins such as GPCRs in E. coli by significantly increasing the expression of various types of GPCR proteins in cell-free systems or E. coli.
도 1은 본 발명의 일 실시예에서 RBS 계산기를 사용하여 다양한 후각 수용체의 번역 초기속도를 예측한 결과 그래프이다.
도 2는 본 발명의 일 실시예에서 RBS 계산기를 사용하여 번역 초기속도에 따른 후각 수용체의 개수를 예측한 결과 그래프이다.
도 3은 본 발명의 일 실시예에서 AT 태그를 삽입한 발현벡터의 구조를 나타낸 모식도이다.
도 4는 본 발명의 일 실시예에서 RBS 계산기를 사용하여 AT 태그에 의한 후각 수용체의 번역 개시율을 예측한 결과 그래프이다.
도 5는 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT 태그에 의한 OR1L4의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면(A) 및 밴드의 강도를 나타낸 그래프(B)이다.
도 6은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT 태그에 의한 OR13C4의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면(A) 및 밴드의 강도를 나타낸 그래프(B)이다.
도 7은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT 태그에 의한 OR2B11의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면(A) 및 밴드의 강도를 나타낸 그래프(B)이다.
도 8은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT 태그에 의한 OR3A3의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면(A) 및 밴드의 강도를 나타낸 그래프(B)이다.
도 9는 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT10 및 AT11 태그에 의한 후각 수용체의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 10은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT10 태그에 의한 후각 수용체의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 11은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT10 태그에 의한, 비슷한 코돈 사용빈도를 갖는 후각 수용체의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 12는 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT10 태그에 의한, 동일한 코돈 사용빈도를 갖는 후각 수용체의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 13은 본 발명의 일 실시예에서 무세포 시스템을 사용하여 AT10 태그에 의한 GPCR의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 14는 본 발명의 일 실시예에서 대장균에서 AT10 태그에 의한 GPCR의 발현 변화를 웨스턴 블롯으로 확인한 결과 도면이다.
도 15는 본 발명의 일 실시예에서, AT10 태그에 의한 GPCR의 활성 변화를 형광 소광 분석법으로 확인한 결과 그래프이다.Figure 1 is a graph showing the results of predicting the initial translation speed of various olfactory receptors using the RBS calculator in one embodiment of the present invention.
Figure 2 is a graph showing the results of predicting the number of olfactory receptors according to the initial translation speed using the RBS calculator in one embodiment of the present invention.
Figure 3 is a schematic diagram showing the structure of an expression vector into which an AT tag is inserted in one embodiment of the present invention.
Figure 4 is a graph showing the results of predicting the translation initiation rate of olfactory receptors by AT tags using an RBS calculator in one embodiment of the present invention.
Figure 5 is a graph (A) showing the results of confirming the expression change of OR1L4 by AT tag by Western blot using a cell-free system in an example of the present invention and the intensity of the band (B).
Figure 6 is a diagram (A) showing the results of confirming the expression change of OR13C4 by AT tag by Western blot using a cell-free system in an example of the present invention and a graph (B) showing the intensity of the band.
Figure 7 is a graph (A) showing the results of confirming the expression change of OR2B11 by AT tag by Western blot using a cell-free system in an example of the present invention and the intensity of the band (B).
Figure 8 is a graph (A) showing the results of confirming the expression change of OR3A3 by AT tag by Western blot using a cell-free system in an example of the present invention and the intensity of the band (B).
Figure 9 is a diagram showing the results of Western blot confirmation of changes in expression of olfactory receptors due to AT10 and AT11 tags using a cell-free system in one embodiment of the present invention.
Figure 10 is a diagram showing the results of Western blot confirmation of changes in the expression of olfactory receptors due to AT10 tags using a cell-free system in one embodiment of the present invention.
Figure 11 is a diagram showing the results of Western blot confirmation of changes in expression of olfactory receptors with similar codon usage frequencies due to AT10 tags using a cell-free system in one embodiment of the present invention.
Figure 12 is a diagram showing the results of Western blot confirmation of changes in expression of olfactory receptors with the same codon usage frequency due to the AT10 tag using a cell-free system in one embodiment of the present invention.
Figure 13 is a diagram showing the results of confirming the change in GPCR expression due to the AT10 tag by Western blot using a cell-free system in one embodiment of the present invention.
Figure 14 is a diagram showing the results of confirming the expression change of GPCR due to AT10 tag in E. coli by Western blot in one embodiment of the present invention.
Figure 15 is a graph showing the results of confirming the change in GPCR activity due to the AT10 tag using fluorescence quenching analysis in one embodiment of the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 아데닌(adenine, A) 및 티민(thymine, T)으로 구성되고, 3 내지 99개의 뉴클레오타이드로 구성되는 막 단백질(membrane protein) 발현 증진용 태그(tag)를 제공한다.The present invention provides a tag for enhancing the expression of a membrane protein consisting of adenine (A) and thymine (T) and 3 to 99 nucleotides.
상기 태그는 아데닌 및 티민으로 구성되어 리신(lysine, K) 또는 티로신(tyrosine, Y)을 암호화할 수 있다. 즉, 상기 뉴클레오타이드는 AAA 또는 TAT의 코돈으로 구성되고, 3의 배수로 포함될 수 있다. 상기 뉴클레오타이드는 3 내지 99개, 3 내지 66개, 3 내지 33개, 6 내지 99개, 6 내지 66개, 6 내지 33개, 9 내지 99개, 9 내지 66개, 9 내지 33개, 12 내지 99개, 12 내지 66개, 12 내지 33개, 15 내지 99개, 15 내지 66개, 15 내지 33개, 18 내지 99개, 18 내지 66개, 18 내지 33개, 21 내지 99개, 21 내지 66개, 21 내지 33개, 24 내지 99개, 24 내지 66개, 24 내지 33개, 27 내지 99개, 27 내지 66개, 27 내지 33개, 30 내지 99개, 30 내지 66개 또는 30 내지 33개의 뉴클레오타이드로 구성될 수 있다.The tag is composed of adenine and thymine and can encode lysine (K) or tyrosine (Y). That is, the nucleotides are composed of codons of AAA or TAT and may be included in multiples of 3. The nucleotides are 3 to 99, 3 to 66, 3 to 33, 6 to 99, 6 to 66, 6 to 33, 9 to 99, 9 to 66, 9 to 33, 12 to 99, 12 to 66, 12 to 33, 15 to 99, 15 to 66, 15 to 33, 18 to 99, 18 to 66, 18 to 33, 21 to 99, 21 to 66, 21 to 33, 24 to 99, 24 to 66, 24 to 33, 27 to 99, 27 to 66, 27 to 33, 30 to 99, 30 to 66 or 30 to It can consist of 33 nucleotides.
본 발명의 다른 측면에서, 상기 뉴클레오타이드에 의해 번역되는 폴리펩타이드는 1 내지 33개, 1 내지 22개, 1 내지 11개, 2 내지 33개, 2 내지 22개, 2 내지 11개, 3 내지 33개, 3 내지 22개, 3 내지 11개, 4 내지 33개, 4 내지 22개, 4 내지 11개, 5 내지 33개, 5 내지 22개, 5 내지 11개, 6 내지 33개, 6 내지 22개, 6 내지 11개, 7 내지 33개, 7 내지 22개, 7 내지 11개, 8 내지 33개, 8 내지 22개, 8 내지 11개, 9 내지 33개, 9 내지 22개, 9 내지 11개, 10 내지 33개, 10 내지 22개 또는 10 내지 11개의 아미노산으로 구성된 폴리펩타이드일 수 있다.In another aspect of the invention, the polypeptide translated by the nucleotide is 1 to 33, 1 to 22, 1 to 11, 2 to 33, 2 to 22, 2 to 11, 3 to 33. , 3 to 22, 3 to 11, 4 to 33, 4 to 22, 4 to 11, 5 to 33, 5 to 22, 5 to 11, 6 to 33, 6 to 22 , 6 to 11, 7 to 33, 7 to 22, 7 to 11, 8 to 33, 8 to 22, 8 to 11, 9 to 33, 9 to 22, 9 to 11 , may be a polypeptide consisting of 10 to 33, 10 to 22, or 10 to 11 amino acids.
이때, 상기 태그는 이에 의해 번역되는 첫번째 아미노산의 C-말단으로부터 KYY가 순차적으로 하나씩 추가된 것일 수 있다. 구체적으로, 상기 태그는 첫번째 아미노산으로 K를, 두번째 아미노산으로 Y를, 세번째 아미노산으로 Y를 포함하며, 아미노산의 수가 증가할 때마다 이의 C-말단에 KYY가 순차적으로 하나씩 추가된 것일 수 있다. 예를 들어, 상기 태그가 하나의 아미노산으로 구성되는 경우 K, 두개의 아미노산으로 구성되는 경우 KY, 세개의 아미노산으로 구성되는 경우 KYY, 네개의 아미노산으로 구성되는 경우 KYYK, 다섯개의 아미노산으로 구성되는 경우 KYYKY, 여섯개의 아미노산으로 구성되는 경우 KYYKYY의 서열로 표시되는 폴리펩타이드를 의미하는 것일 수 있다. 즉, 본 발명에 따른 태그는, 하기 표 1에 나타난 바와 같이, 이에 의해 번역되는 폴리펩타이드가 서열번호 51 내지 83으로 기재된 아미노산 서열로 구성된 군으로부터 선택되는 폴리펩타이드일 수 있다. 본 발명의 일 실시예에서, 상기 폴리펩타이드는 서열번호 51 내지 61으로 기재된 아미노산 서열로 구성된 군으로부터 선택되는 폴리펩타이드일 수 있다.At this time, the tag may be one in which KYY is sequentially added from the C-terminus of the first amino acid translated thereby. Specifically, the tag includes K as the first amino acid, Y as the second amino acid, and Y as the third amino acid, and each time the number of amino acids increases, KYY may be sequentially added to the C-terminus. For example, if the tag consists of one amino acid, K, if it consists of two amino acids, KY, if it consists of three amino acids, KYY, if it consists of four amino acids, KYYK, if it consists of five amino acids, KYYKY, when composed of six amino acids, may refer to a polypeptide represented by the sequence of KYYKYY. That is, as shown in Table 1 below, the tag according to the present invention may be a polypeptide translated by the tag selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 51 to 83. In one embodiment of the present invention, the polypeptide may be a polypeptide selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 51 to 61.
상기 용어, "막 단백질(membrane protein)"은 생체막의 일부분이거나 생체막과 상호작용하는 단백질을 의미한다. 상기 막 단백질은 막의 일부분이거나 막에 영구적으로 부착된 내재성 막 단백질과 일시적으로 인지질 이중층 또는 다른 내재성 막 단백질에 부착된 내재성 단일체 단백질(integral monotopic protein)로 구분된다. 구체적으로, 본 발명에 따른 단백질은 내재성 막 단백질을 의미하는 것일 수 있다.The term “membrane protein” refers to a protein that is part of or interacts with a biological membrane. The membrane proteins are divided into integral membrane proteins that are part of the membrane or permanently attached to the membrane, and integral monotopic proteins that are temporarily attached to the phospholipid bilayer or other integral membrane proteins. Specifically, the protein according to the present invention may refer to an integral membrane protein.
상기 용어, "내재성 막 단백질(integral membrane protein)"은 생물학적 막에 영구적으로 부착되는 막 단백질의 하나로서, 예로서 수송체, 링커, 채널, 수용체, 효소, 구조적 막 고정 도메인, 에너지 축적 및 변환에 관여하는 단백질, 세포 부착에 관여하는 단백질 등이 포함된다. 구체적으로, 상기 내재성 막 단백질은 G 단백질 결합 수용체(G protein-coupled receptor, GPCR), 호르몬 수용체(hormone receptor), 세포막 수송 단백질 (membrane transport protein), 세포 접착 분자(cell adhesion molecule), 효소 연결 수용체(enzyme-linked receptor) 등을 포함할 수 있다.The term "integral membrane protein" refers to any of the membrane proteins permanently attached to biological membranes, such as transporters, linkers, channels, receptors, enzymes, structural membrane anchoring domains, energy accumulation and transduction. Includes proteins involved in , proteins involved in cell adhesion, etc. Specifically, the integral membrane proteins include G protein-coupled receptors (GPCR), hormone receptors, membrane transport proteins, cell adhesion molecules, and enzyme linkages. It may include receptors (enzyme-linked receptors), etc.
상기 용어, "G 단백질 결합 수용체(G protein-coupled receptor, GPCR)"는 7-막관통 수용체(7-transmembrane receptor, 7TMR)라고도 불리는 단백질로서, 세포막을 7번 통과하는 구조를 갖는 단백질을 의미한다. 상기 GPCR은 생체 내의 거의 모든 생리적 반응을 촉매하며, 이에 결합하는 물질을 리간드라 부른다. 상기 리간드는 수용체의 반응을 촉진하는 수용체 작용제와 반응을 촉진하지 않는 수용체 대항체로 구분할 수 있다. 예를 들어, 상기 GPCR은 클래스 A, 클래스 B, 클래스 C, 클래스 D, 클래스 E 또는 클래스 F의 GPCR로 분류할 수 있다.The term "G protein-coupled receptor (GPCR)" is also called a 7-transmembrane receptor (7TMR) and refers to a protein that has a structure that passes through the cell membrane seven times. . The GPCR catalyzes almost all physiological reactions in the living body, and the substance that binds to it is called a ligand. The ligand can be divided into receptor agonists that promote the receptor response and receptor antagonists that do not promote the reaction. For example, the GPCRs can be classified as class A, class B, class C, class D, class E, or class F GPCRs.
예를 들어, 상기 클래스 A에 포함되는 GPCR은 후각 수용체(olfactory receptor, OR), CC 케모카인 수용체(CC chemokine receptor, CCR), CXC 케모카인 수용체(CXC chemokine receptor, CXCR), 아데노신 A2a 수용체(adenosine A2a receptor, ADORA), B형 엔도세린 수용체(endothelin receptor type B, ENDRB), 아펠린 수용체(apelin receptor, APLNR) 등이 있다. 한편, 클래스 B에 포함되는 GPCR은 PTH/PTHrP 수용체(PTH/PTHrP receptor, PTHR) 등이 있으며, 클래스 C에 포함되는 GPCR은 미각 수용체(taste receptor) 등이 있다.For example, GPCRs included in class A include olfactory receptor (OR), CC chemokine receptor (CCR), CXC chemokine receptor (CXCR), and adenosine A2a receptor. , ADORA), endothelin receptor type B (ENDRB), and apelin receptor (APLNR). Meanwhile, GPCRs included in class B include PTH/PTHrP receptor (PTHR), and GPCRs included in class C include taste receptors.
상기 용어, "호르몬 수용체(hormone receptor)"는 특정 호르몬과 결합하여 이를 세포에 전달하는 단백질로서, 세포막을 통과하지 못하는 호르몬에 의한 세포 밖 신호를 세포 안의 신호로 전달하는 역할을 한다. 상기 호르몬 수용체는 G-단백질 연관 수용체, 단백질 인산화 수용체, 이온통로 수용체 등을 포함할 수 있다.The term "hormone receptor" refers to a protein that binds to a specific hormone and transmits it to a cell. It serves to transmit an extracellular signal caused by a hormone that does not pass through the cell membrane into an intracellular signal. The hormone receptors may include G-protein-related receptors, protein phosphorylation receptors, ion channel receptors, etc.
상기 용어, "세포막 수송 단백질(membrane transport protein)"은 생체 막을 가로지르는 이온 및 저분자나, 다른 단백질과 같은 거대 분자의 이동에 관여하는 단백질로서, 수송체(transporter)라고도 불린다. 상기 세포막 수송 단백질은 촉진 확산이나 능동 수송을 통해 물질을 이동시키며, 통로(channel) 또는 운반체(carrier)의 두 가지 유형으로 광범위하게 분류될 수 있다.The term "membrane transport protein" refers to a protein involved in the movement of ions, small molecules, or macromolecules such as other proteins across biological membranes, and is also called a transporter. The cell membrane transport proteins move substances through facilitated diffusion or active transport and can be broadly classified into two types: channels or carriers.
상기 용어, "세포 접착 분자(cell adhesion molecule)"는 대부분 막 관통형 당단백질로서 당사슬에 의해 막과 유착된 형태로 존재하는 단백질로서, 세포의 발생에서 조직형성 과정, 면역시스템 내에서 세포간 상호작용, 종양의 침윤성 등에 관여하는 것으로 알려진 단백질을 의미한다.The term "cell adhesion molecule" refers to a protein that is mostly transmembrane glycoprotein and exists in the form of adhesion to the membrane by sugar chains, and is used during cell development, tissue formation, and intercellular interactions within the immune system. It refers to a protein known to be involved in the action, tumor invasiveness, etc.
상기 용어, "효소 연결 수용체(enzyme-linked receptor)"는 효소의 활성을 이용하여 신호전달 경로를 개시하는 수용체로서, 자신의 세포질 영역에 효소 활성으로서 티로신 인산화효소 활성, 세린-트레오닌 인산화효소 활성, 구아닐산 고리화효소 활성을 갖거나, 자신은 효소활성 부위는 없지만 세포질 영역에서 효소활성을 갖춘 분자와 상호작용하는 수용체로 분류될 수 있다. 예로서, 상기 효소 연결 수용체는 톨-유사 수용체(toll-like receptor, TLR), 교세포 신경성장인자(glial cell-derived neurotrophic factor, GDNF), 심방나트륨이뇨펩티드 수용체(atrial natriuretic peptide receptor, ANPR) 등을 포함할 수 있다.The term "enzyme-linked receptor" refers to a receptor that initiates a signal transduction pathway using the activity of an enzyme, and the enzyme activity in its cytoplasmic region includes tyrosine phosphatase activity, serine-threonine phosphatase activity, It can be classified as a receptor that has guanylate cyclase activity or that has no enzymatic activity site itself but interacts with molecules that possess enzymatic activity in the cytoplasmic region. For example, the enzyme-linked receptors include toll-like receptor (TLR), glial cell-derived neurotrophic factor (GDNF), atrial natriuretic peptide receptor (ANPR), etc. may include.
본 발명에 따른 막 단백질 발현 증진용 태그는 발현시키고자 하는 막 단백질의 초기 발현 속도를 증진시키는 것일 수 있다.The tag for enhancing membrane protein expression according to the present invention may enhance the initial expression rate of the membrane protein to be expressed.
또한, 본 발명은 상기 막 단백질 발현 증진용 태그가 포함된 발현벡터를 제공한다.Additionally, the present invention provides an expression vector containing a tag for enhancing expression of the membrane protein.
본 발명에 따른 발현벡터에 포함된 막 단백질 발현 증진용 태그는 상기 서술한 바와 같은 특징을 가질 수 있다. 이때, 상기 막 단백질 발현 증진용 태그는 발현시키고자 하는 막 단백질의 시작 코돈 뒤에 삽입될 수 있다.The tag for enhancing membrane protein expression included in the expression vector according to the present invention may have the characteristics described above. At this time, the tag for enhancing membrane protein expression may be inserted after the start codon of the membrane protein to be expressed.
상기 용어, "발현벡터(expression vector)"는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터, 코스미드 백터, 박테리오파지 벡터, 바이러스 벡터 등을 모두 포함할 수 있다. 상기 발현벡터는 이에 포함되는 핵산으로부터 펩티드를 생성하기 위해 필요한 요소를 포함할 수 있다. 구체적으로, 상기 발현벡터는 신호서열, 복제기점, 마커 유전자, 프로모터, 전사 종결 서열 등을 포함할 수 있다. 이때, 본 발명에 따른 항체 또는 이의 항원 결합 단편이 암호화된 핵산은 프로모터와 작동적으로 연결될 수 있다.The term “expression vector” refers to a means for expressing a target gene in a host cell and may include plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. The expression vector may contain elements necessary for producing a peptide from the nucleic acid contained therein. Specifically, the expression vector may include a signal sequence, origin of replication, marker gene, promoter, transcription termination sequence, etc. At this time, the nucleic acid encoding the antibody or antigen-binding fragment thereof according to the present invention may be operably linked to a promoter.
일례로, 원핵세포에 사용되는 발현벡터는 전사를 진행시키는 프로모터, 해독의 개시를 위한 라이보좀 결합 자리, 및 전사와 해독의 종결서열을 포함할 수 있다. 한편, 진핵세포에 사용되는 발현벡터는 포유동물 또는 포유동물 바이러스로부터 유래된 프로모터 및 폴리아데닐화 서열을 포함할 수 있다.For example, an expression vector used in prokaryotic cells may include a promoter for transcription, a ribosome binding site for initiation of translation, and termination sequences for transcription and translation. Meanwhile, expression vectors used in eukaryotic cells may include a promoter and polyadenylation sequence derived from a mammal or a mammalian virus.
또한, 상기 발현벡터에 포함되는 마커 유전자로는 통상의 기술분야에 알려진 것을 모두 사용할 수 있으며, 구체적으로 항생제 내성 유전자일 수 있다. 구체적으로, 상기 항생제 내성 유전자는 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 네오마이신, 테트라사이클린 등을 포함하는 항생제에 대해 내성을 나타내는 유전자일 수 있다. Additionally, any marker gene included in the expression vector can be any one known in the art, and specifically, it may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene expressing resistance to antibiotics including ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, etc.
또한, 본 발명은 상기 발현벡터가 형질전환된 숙주세포를 제공한다.Additionally, the present invention provides host cells transformed with the expression vector.
본 발명에 따른 숙주세포에 형질전환된 재조합 벡터는 상술한 바와 같은 특징을 가질 수 있다. 상기 숙주세포는 상술한 바와 같은 특징을 갖는 재조합 벡터가 형질전환됨으로써 재조합 벡터에 의해 유전적으로 변형될 수 있다.The recombinant vector transformed into a host cell according to the present invention may have the characteristics described above. The host cell can be genetically modified by a recombinant vector by being transformed with a recombinant vector having the characteristics described above.
상기 숙주세포는 통상의 기술분야에 재조합 단백질, 구체적으로 재조합 막 단백질을 생산하기 위해 사용될 수 있다고 알려진 모든 종류의 세포를 사용할 수 있다. 구체적으로, 상기 숙주세포는 원핵세포, 효모 또는 진핵세포일 수 있다. 상기 원핵세포는 대장균(E. coli), 바실러스 속 균주, 스트렙토마이세스 속 균주, 슈도모나스 속 균주, 스타필로코쿠스 속 균주 등을 포함할 수 있고, 상기 효모는 사카로마이세스 세레비지애 등을 포함할 수 있다. 한편, 상기 진핵세포는 COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PERC6, SP2/0, NS-0, U20S 및 HT1080 등을 포함할 수 있다.The host cell may be any type of cell known in the art that can be used to produce recombinant proteins, specifically recombinant membrane proteins. Specifically, the host cell may be a prokaryotic cell, yeast, or eukaryotic cell. The prokaryotic cells may include E. coli , Bacillus genus strains, Streptomyces genus strains, Pseudomonas genus strains, Staphylococcus genus strains, etc., and the yeast includes Saccharomyces cerevisiae, etc. It can be included. Meanwhile, the eukaryotic cells include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, It may include W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PERC6, SP2/0, NS-0, U20S and HT1080.
또한, 상기 숙주세포는 통상의 기술분야에 알려진 방법에 따라 상술한 바와 같은 발현벡터가 형질주입된 것일 수 있다. 구체적으로, 상기 형질주입은 일시적 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개 형질감염(liposome-mediated transfection), DEAE 덱스트란-매개 형질감염(DEAE dextran-mediated transfection), 폴리브렌-매개 형질감염(polybrene-mediated transfection), 전기침공법, 유전자 총(gene gun) 등의 방법으로 수행될 수 있다. 또한, 상기 방법은 통상의 기술자에 의해 적절히 변형될 수 있다.Additionally, the host cell may have been transfected with the expression vector described above according to a method known in the art. Specifically, the transfection includes transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, and DEAE dextran-mediated transfection. It can be performed by methods such as (DEAE dextran-mediated transfection), polybrene-mediated transfection, electroporation, and gene gun. Additionally, the method may be appropriately modified by those skilled in the art.
나아가, 본 발명은 상기 숙주세포에서 막 단백질을 발현시키는 단계를 포함하는 막 단백질의 발현을 증가시키는 방법을 제공한다.Furthermore, the present invention provides a method for increasing the expression of a membrane protein comprising expressing the membrane protein in the host cell.
본 발명에 따른 방법으로 생산되는 상기 서술한 바와 같은 태그를 포함하는 막 단백질일 수 있다. 상기 막 단백질의 발현 증가는 단백질의 초기 발현 속도 증가에 의한 것일 수 있다.It may be a membrane protein containing a tag as described above produced by the method according to the present invention. The increased expression of the membrane protein may be due to an increase in the initial expression rate of the protein.
상기 막 단백질은 숙주세포를 배양함으로써 발현될 수 있다. 상기 배양은 막 단백질의 생산에 사용되는 숙주세포의 종류에 따라 적절한 배양배지를 사용하여 수행될 수 있고, 필요에 따라 적절한 보충물이 포함될 수 있다. 또한, 상기 배양은 숙주세포의 종류에 따라 적절한 환경에서 수행될 수 있다.The membrane protein can be expressed by culturing host cells. The culture may be performed using an appropriate culture medium depending on the type of host cell used to produce the membrane protein, and appropriate supplements may be included as needed. Additionally, the culture may be performed in an appropriate environment depending on the type of host cell.
본 발명에 따른 생산 방법은 숙주세포에서 생산된 막 단백질을 회수하는 단계를 더 포함할 수 있다. 상기 회수는 통상의 기술분야에 알려진 방법에 따라 수행될 수 있으며, 이는 필요에 따라 통상의 기술자에 의해 적절히 변형될 수 있다. 일례로, 상기 회수는 원심분리 또는 한외여과를 이용하여 불순물을 제거하고, 수득된 결과물을 크로마토그래피 등으로 추가 정제함으로써 수행될 수 있다. 상기 크로마토그래피는 친화 크로마토그래피, 양이온 크로마토그래피, 소수성 상호작용 크로마토그래피 등을 포함할 수 있다.The production method according to the present invention may further include the step of recovering the membrane protein produced in the host cell. The recovery may be performed according to methods known in the art, which may be appropriately modified by those skilled in the art as necessary. For example, the recovery may be performed by removing impurities using centrifugation or ultrafiltration and further purifying the obtained result using chromatography or the like. The chromatography may include affinity chromatography, cation chromatography, hydrophobic interaction chromatography, etc.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다, 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 이들에 의해 본 발명이 제한되는 것은 아니다. 본 발명의 청구범위에 기재된 기술적 사상과 실질적으로 동일한 구성을 갖고 동일한 작용 효과를 이루는 것은 어떠한 것이라도 본 발명의 기술적 범위에 포함된다.Hereinafter, the present invention will be described in detail through the following examples. However, the following examples are only for illustrating the present invention and are not intended to limit the present invention thereto. Anything that has substantially the same structure as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.
실시예 1. 인간 후각 수용체의 번역 초기속도 예측Example 1. Prediction of initial translation rate of human olfactory receptors
약 400종의 인간 후각 수용체(olfactory receptor)를 사용하여 번역(translation) 초기속도를 통상적인 방법으로 RBS 계산기(RBS calculator, https://salislab.net/software/predict_rbs_calculator)로 예측하였다. 예측된 결과를 이용하여 후각 수용체의 종류에 따른 번역 개시율(initiation rate)을 도 1에, 각 번역 초기속도에 해당하는 후각 수용체 개수를 도 2에 나타내었다.Using about 400 types of human olfactory receptors, the initial translation speed was predicted using the RBS calculator (RBS calculator, https://salislab.net/software/predict_rbs_calculator) using a conventional method. Using the predicted results, the translation initiation rate according to the type of olfactory receptor is shown in Figure 1, and the number of olfactory receptors corresponding to each initial translation rate is shown in Figure 2.
도 1에 나타난 바와 같이, 인간 후각 수용체의 번역 개시율은 다양한 분포를 보이며, 도 2에 나타난 바와 같이 대다수의 인간 후각 수용체가 낮은 번역 초기속도를 보였다.As shown in Figure 1, the translation initiation rate of human olfactory receptors shows a diverse distribution, and as shown in Figure 2, the majority of human olfactory receptors showed a low translation initiation rate.
실시예 2. AT 태그에 의한 후각 수용체의 번역 초기속도 예측Example 2. Prediction of initial translation rate of olfactory receptor by AT tag
인간 후각 수용체를 암호화하는 염기서열의 개시코돈 다음에 AT 태그를 삽입(도 3)한 경우의 번역 초기속도를 상기와 같은 방법으로 예측하였다. 이때, 인간 후각 수용체는 OR1D2(olfactory receptor family 1 subfamily D member 2), OR1L4(olfactory receptor family 1 subfamily L member 2), OR2B11(olfactory receptor family 2 subfamily B member 11), OR3A3(olfactory receptor family 3 subfamily A member 3), OR4F16(olfactory receptor family 4 subfamily F member 16), OR6P1(olfactory receptor family 6 subfamily P member 1), OR9A2(olfactory receptor family 9 subfamily A member 2) 또는 OR13C4(olfactory receptor family 13 subfamily C member 4)를 사용하였고, AT 태그는 하기 표 2에 기재된 바와 같이 사용하였다. 예측된 결과를 이용하여, AT 태그의 종류에 따른 번역 개시율을 도 4에 나타내었다.The initial translation speed when an AT tag was inserted after the start codon of the base sequence encoding the human olfactory receptor (Figure 3) was predicted using the same method as above. At this time, the human olfactory receptors are OR1D2 (olfactory receptor family 1 subfamily D member 2), OR1L4 (olfactory receptor family 1 subfamily L member 2), OR2B11 (olfactory receptor family 2 subfamily B member 11), and OR3A3 (olfactory receptor family 3 subfamily A). member 3), OR4F16 (olfactory receptor family 4 subfamily F member 16), OR6P1 (olfactory receptor family 6 subfamily P member 1), OR9A2 (olfactory receptor family 9 subfamily A member 2), or OR13C4 (olfactory receptor family 13 subfamily C member 4) ) was used, and the AT tag was used as described in Table 2 below. Using the predicted results, the translation initiation rate according to the type of AT tag is shown in Figure 4.
도 4에 나타난 바와 같이, AT1 내지 AT8 태그의 경우, 후각 수용체의 종류에 따라 상이한 번역 개시율을 나타낸 반면, AT9, AT10 및 AT11 태그는 후각 수용체의 종류와 무관하게 일관되고 빠른 번역 개시율을 보였다.As shown in Figure 4, AT1 to AT8 tags showed different translation initiation rates depending on the type of olfactory receptor, while AT9, AT10, and AT11 tags showed consistent and fast translation initiation rates regardless of the type of olfactory receptor. .
실시예 3. AT 태그가 삽입된 후각 수용체를 포함하는 발현벡터의 제작Example 3. Construction of an expression vector containing an AT tag-inserted olfactory receptor
상기 실시예에서 프로그램을 사용하여 확인된 번역 개시율을 실험을 통해 재확인하고자 AT 태그가 삽입된 후각 수용체를 포함하는 발현벡터를 다음과 같은 방법으로 제작하였다. 이때, 후각 수용체는 OR1L4, OR13C4, OR2B11 또는 OR3A3를 사용하였다.In order to reconfirm through experiment the translation initiation rate confirmed using the program in the above example, an expression vector containing an olfactory receptor with an AT tag inserted was produced in the following manner. At this time, OR1L4, OR13C4, OR2B11, or OR3A3 were used as olfactory receptors.
먼저, 후각 수용체인 OR1L4, OR13C4, OR2B11 또는 OR3A3를 암호화하는 유전자를 게이트웨이™ 엔트리(Gateway™ entry) 클로닝 방법을 사용하여 대장균 발현벡터인 pET-DEST42 벡터에 클로닝하였다. 이후, 후각 수용체가 클로닝된 pET-DEST42 벡터를 하기 표 3에 기재된 프라이머를 사용하여 증폭하였다.First, genes encoding olfactory receptors OR1L4, OR13C4, OR2B11, or OR3A3 were cloned into the pET-DEST42 vector, an E. coli expression vector, using the Gateway™ entry cloning method. Afterwards, the pET-DEST42 vector in which the olfactory receptor was cloned was amplified using the primers listed in Table 3 below.
증폭된 PCR 산물은 AccuPrep® PCR/겔 DNA 정제 키트(AccuPrep® PCR/Gel purification kit)를 사용하여 정제하고, DpnI, T4 폴리뉴클레오타이드 카이네이즈(polynucleotide kinase) 및 T4 라이게이즈(ligase)를 순차적으로 반응시켜 순도 높은 발현벡터를 제작하였다. 제작된 발현벡터를 통상적인 방법으로 DH5α 수용성 세포(competent cell)에 형질전환시킨 후, 암피실린에 내성을 나타내는 콜로니를 선택 및 배양하였다. 라보패스™ 플라스미드 DNA 정제 키트(LaboPass™ plasmid DNA purification kit)를 사용하여 상기 배양된 세포로부터 발현벡터를 추출하고 염기서열 분석을 통해 발현벡터 내 AT 태그가 삽입된 후각 수용체를 확인하였다.The amplified PCR product was purified using AccuPrep ® PCR/Gel DNA purification kit, and sequentially reacted with DpnI, T4 polynucleotide kinase, and T4 ligase . A high purity expression vector was produced. The constructed expression vector was transformed into DH5α competent cells using a conventional method, and then colonies showing resistance to ampicillin were selected and cultured. The expression vector was extracted from the cultured cells using the LaboPass™ plasmid DNA purification kit, and the olfactory receptor into which the AT tag was inserted in the expression vector was confirmed through base sequence analysis.
실시예 4. 후각 수용체의 단백질 발현 확인Example 4. Confirmation of protein expression of olfactory receptors
상기 제작된 플라스미드를 이용하여 무세포 단백질 합성을 수행함으로써, AT 태그에 의한 후각 수용체 단백질의 발현을 다음과 같이 확인하였다.By performing cell-free protein synthesis using the above-constructed plasmid, the expression of olfactory receptor protein by AT tag was confirmed as follows.
4-1. 세포 추출액의 제조4-1. Preparation of cell extract
먼저, 무세포 단백질 합성에 사용하기 위한 세포 추출액을 제조하여 준비하였다.First, a cell extract for use in cell-free protein synthesis was prepared and prepared.
구체적으로, BL21(DE3) 균주를 통상적으로 배양하여 OD600의 값이 0.6이 되었을 때, 1 mM의 IPTG를 첨가하여 T7 RNA 중합효소의 과발현을 유도하였다. 이후, OD600 값이 0.3이 되었을 때, 배양액을 4,500 rpm 및 4℃의 조건에서 15분 동안 원심분리하여 세포를 수집하였다. 수집된 세포에 완충액 A(10 mM 트리스-아세테이트 (pH 8.2), 14 mM 아세트산마그네슘, 60 mM 글루탐산칼륨(potassium glutamate) 및 1 mM DDT(dithiothreitol))를 첨가하여 세척하였고. 세척은 총 3회 반복하였다. 세척된 세포 1 g당 1 ㎖의 완충액 A를 첨가하고 세포를 현탁시켰다. 세포 현탁액을 20 ㎑의 주파수 및 50% 진폭에서 3.175 ㎜ 직경 프로브를 이용하여 767 J의 에너지로 초음파 처리하여 세포를 파쇄하였다(Q125 sonicator, Qsonica). 상기 수득된 세포 파쇄액을 18,000 ×g 및 4℃의 조건에서 30분 동안 원심분리하여 상등액을 회수하였다. 수득된 상등액은 37℃에서 1시간 동안 교반하고 소량으로 분주하여 사용전까지 -80℃에서 보관하였다.Specifically, the BL21(DE3) strain was cultured normally, and when the OD 600 value reached 0.6, 1 mM IPTG was added to induce overexpression of T7 RNA polymerase. Afterwards, when the OD 600 value reached 0.3, the culture medium was centrifuged at 4,500 rpm and 4°C for 15 minutes to collect cells. The collected cells were washed by adding buffer A (10mM Tris-acetate (pH 8.2), 14mM magnesium acetate, 60mM potassium glutamate and 1mM DDT (dithiothreitol)). Washing was repeated a total of three times. 1 ml of buffer A was added per 1 g of washed cells and the cells were suspended. The cell suspension was sonicated at an energy of 767 J using a 3.175 mm diameter probe at a frequency of 20 kHz and 50% amplitude to disrupt the cells (Q125 sonicator, Qsonica). The obtained cell lysate was centrifuged at 18,000 × g and 4°C for 30 minutes to recover the supernatant. The obtained supernatant was stirred at 37°C for 1 hour, dispensed into small portions, and stored at -80°C until use.
4-2. 무세포 단백질 합성4-2. Cell-free protein synthesis
무세포 단백질 합성을 위해, 먼저 57 mM의 HEPES-KOH(pH 8.2), 1.2 mM의 ATP, 0.85 mM의 CTP, 0.85 mM의 GTP, 0.85 mM의 UTP, 170 ㎍/㎖의 대장균 tRNA 혼합물, 2 mM의 각 아미노산들, 33 mM의 포스포엔올피루브산(phosphoenolpyruvate), 1 mM의 DTT, 130 mM의 아세트산칼륨(potassium acetate), 12 mM의 아세트산마그네슘(magnesium acetate), 34 ㎍/㎖의 폴리닌산(folinic acid), 2%(w/v)의 PEG(8000), 13.3 ㎍/㎖의 후각 수용체 발현벡터 및 27%(v/v)의 실시예 4-1에서 제조된 세포 추출액을 혼합하여 반응액을 제조하였다. 제조된 반응액을 2 ㎖의 소형 시험관에 넣고, 30℃의 항온수조에서 4시간 동안 반응시켰다. 반응이 끝난 후, 이를 18,000 ×g 및 4℃의 조건에서 30분 동안 원심분리하여 상층액을 제거하였다. 수득된 펠렛(pellet)에 가용 완충액(20 mM Tris-HCl(pH 8.0), 20 mM SDS, 100 mM DTT, 1 mM EDTA)를 첨가하고 37℃에서 1시간 동안 반응시켰다. 상기 가용화된 반응물을 사용하여 웨스턴 블롯을 통상적인 방법으로 수행함으로써 단백질의 발현 정도를 확인하였다. 이때, 1차 항체로서 V5 태그에 결합하는 토끼 유래 항체를, 2차 항체로서 토끼 항체에 결합하는 HRP 효소가 결합된 염소 유래 항체를 사용하였다. 그 결과, OR1L4, OR13C4, OR2B11 또는 OR3A3에 대한 웨스턴 블롯 결과를 촬영한 사진 및 발현 정도를 정량한 결과를 각각 도 5 내지 8에 나타내었다.For cell-free protein synthesis, first 57mM HEPES-KOH (pH 8.2), 1.2mM ATP, 0.85mM CTP, 0.85mM GTP, 0.85mM UTP, 170 μg/ml E. coli tRNA mixture, 2mM Each amino acid, 33mM phosphoenolpyruvate, 1mM DTT, 130mM potassium acetate, 12mM magnesium acetate, 34㎍/㎖ polynic acid ( folinic acid), 2% (w/v) of PEG (8000), 13.3 μg/ml of the olfactory receptor expression vector, and 27% (v/v) of the cell extract prepared in Example 4-1 were mixed to create a reaction solution. was manufactured. The prepared reaction solution was placed in a small 2 ml test tube and reacted in a constant temperature water bath at 30°C for 4 hours. After the reaction was completed, it was centrifuged at 18,000 × g and 4°C for 30 minutes to remove the supernatant. Soluble buffer (20mM Tris-HCl (pH 8.0), 20mM SDS, 100mM DTT, 1mM EDTA) was added to the obtained pellet and reacted at 37°C for 1 hour. The level of protein expression was confirmed by performing Western blotting using the solubilized reaction product in a conventional manner. At this time, a rabbit-derived antibody that binds to the V5 tag was used as the primary antibody, and a goat-derived antibody bound to the HRP enzyme that binds to the rabbit antibody was used as the secondary antibody. As a result, photographs of the Western blot results for OR1L4, OR13C4, OR2B11, or OR3A3 and the results of quantifying the expression level are shown in Figures 5 to 8, respectively.
도 5 내지 8에 나타난 바와 같이, 후각 수용체만을 발현시킨 경우와 비교하여 AT 태그가 포함된 후각 수용체의 발현이 유의적으로 증가하였다. 특히, AT10 태그의 경우, 모든 후각 수용체에서 발현 증가 효과가 가장 우수하였다.As shown in Figures 5 to 8, compared to the case where only the olfactory receptor was expressed, the expression of the olfactory receptor containing the AT tag was significantly increased. In particular, in the case of the AT10 tag, the effect of increasing expression was the best in all olfactory receptors.
실시에 5. AT10 및 AT11에 의한 후각 수용체 발현 비교Example 5. Comparison of olfactory receptor expression by AT10 and AT11
상기 실시예에서 유의적으로 후각 수용체의 발현을 증진시키는 것으로 확인된 AT10 및 AT11 태그를 사용하여 13종류의 후각 수용체 발현을 다음과 같이 비교하였다.The expression of 13 types of olfactory receptors was compared as follows using AT10 and AT11 tags, which were confirmed to significantly enhance the expression of olfactory receptors in the above example.
먼저, OR1G1(olfactory receptor family 1 subfamily G member 1), OR1L1(olfactory receptor family 1 subfamily L member 1), OR1L4, OR3A3, OR6F1(olfactory receptor family 6 subfamily F member 1), OR6Y1(olfactory receptor family 6 subfamily Y member 1), OR9A2, OR13C4, OR2B11, OR2J2(olfactory receptor family 2 subfamily J member 2), OR2W1(olfactory receptor family 2 subfamily W member 1), OR51E1(olfactory receptor family 51 subfamily E member 1) 또는 OR4F16을 암호화하는 유전자를 상술한 바와 같이 pET-DEST42 벡터에 클로닝하였다. 이후, 하기 표 4에 기재된 프라이머를 사용하여 상술한 바와 같은 방법으로 PCR을 수행함으로써, 후각 수용체 후각 수용체 개시코돈 뒤에 AT10 태그가 삽입된 발현벡터를 제조하였다.First, OR1G1 (olfactory receptor family 1 subfamily G member 1), OR1L1 (olfactory receptor family 1 subfamily L member 1), OR1L4, OR3A3, OR6F1 (olfactory receptor family 6 subfamily F member 1), OR6Y1 (olfactory receptor family 6 subfamily Y) member 1), OR9A2, OR13C4, OR2B11, OR2J2 (olfactory receptor family 2 subfamily J member 2), OR2W1 (olfactory receptor family 2 subfamily W member 1), OR51E1 (olfactory receptor family 51 subfamily E member 1), or OR4F16. The gene was cloned into the pET-DEST42 vector as described above. Then, PCR was performed in the same manner as described above using the primers listed in Table 4 below to prepare an expression vector in which an AT10 tag was inserted after the olfactory receptor olfactory receptor start codon.
상기 발현벡터를 사용하여 실시예 4에 기재된 바와 동일한 조건 및 방법으로 무세포 단백질 합성을 수행함으로써 후각 수용체의 발현을 확인하였다. 그 결과, 후각 수용체의 발현정도를 확인한 웨스턴 블롯 결과를 도 9에 나타내었다.Expression of olfactory receptors was confirmed by performing cell-free protein synthesis using the above expression vector under the same conditions and methods as described in Example 4. As a result, the Western blot results confirming the expression level of olfactory receptors are shown in Figure 9.
도 9에 나타난 바와 같이, 12종류의 후각 수용체 중, OR1G1, OR1L1, OR1L4, OR3A3, OR6F1, OR6Y1, OR9A2 및 OR13C4는 AT11 태그를 사용한 경우보다 AT10 태그에 의해 발현이 유의적으로 현저히 증가하였다. 반면, OR2B11, OR2J2, OR2W1 및 OR51E1는 AT10 및 AT11 모두 비슷한 정도로 발현이 증가하였다.As shown in Figure 9, among the 12 types of olfactory receptors, the expression of OR1G1, OR1L1, OR1L4, OR3A3, OR6F1, OR6Y1, OR9A2, and OR13C4 was significantly increased by the AT10 tag compared to the case of using the AT11 tag. On the other hand, the expression of OR2B11, OR2J2, OR2W1, and OR51E1 increased to a similar extent in both AT10 and AT11.
실시예 6. AT10 태그에 의한 후각 수용체 발현 증가 확인-(1)Example 6. Confirmation of increased olfactory receptor expression by AT10 tag-(1)
상기에서 4종류의 후각 수용체 발현을 가장 우수하게 증가시키는 것으로 확인된 AT10 태그를 사용하여 17종류의 후각 수용체 발현 증가를 다음과 같이 확인하였다. 실험은 OR1L1, OR1L4, OR1N1, OR2B6, OR2J2, OR2W1, OR3A3, OR4E2, OR4F16, OR6B2, OR6F1, OR9A2, OR10K1, OR13C4, OR13D1, OR51E1 또는 OR52E5를 암호화하는 유전자와 하기 표 5에 기재된 프라이머를 사용한 것을 제외하고는 상기 서술한 바와 같은 방법으로 수행되었다. 그 결과, 후각 수용체의 발현정도를 확인한 웨스턴 블롯 결과를 도 10에 나타내었다.Using the AT10 tag, which was confirmed to best increase the expression of the four types of olfactory receptors above, the increase in the expression of 17 types of olfactory receptors was confirmed as follows. The experiments used genes encoding OR1L1, OR1L4, OR1N1, OR2B6, OR2J2, OR2W1, OR3A3, OR4E2, OR4F16, OR6B2, OR6F1, OR9A2, OR10K1, OR13C4, OR13D1, OR51E1 or OR52E5 and the primers listed in Table 5 below. It was then performed in the same manner as described above. As a result, the Western blot results confirming the expression level of olfactory receptors are shown in Figure 10.
도 10에 나타난 바와 같이, AT10 태그가 포함되지 않은 발현벡터를 사용한 경우와 비교하여 AT10 태그가 삽입된 경우 후각 수용체의 발현이 현저히 증가하였다.As shown in Figure 10, compared to the case of using an expression vector that does not contain the AT10 tag, the expression of the olfactory receptor was significantly increased when the AT10 tag was inserted.
실시예 7. AT10 태그에 의한 후각 수용체 발현 증가 확인-(2)Example 7. Confirmation of increased olfactory receptor expression by AT10 tag-(2)
상기에서 AT10 태그에 의한 발현 증가가 확인된 후각 수용체 중, 대장균에서 코돈 사용 빈도가 상이하면서 번역 초기속도가 유사한 후각 수용체를 사용하여 AT10 태그에 의한 후각 수용체 발현 변화를 확인하였다. 이때, 후각 수용체로는 OR2B6, OR4F16, OR2W1, OR13C4, OR1L1, OR13D1, OR6F1 및 OR6B2를 사용하여 상기 서술한 바와 같이 무세포 단백질 합성방법으로 수행되었으며 AT10 태그 유무에 따른 결과를 비교하여 표 6 및 도 11에 나타내었다.Among the olfactory receptors whose expression was confirmed to be increased by the AT10 tag above, olfactory receptors with different codon usage frequencies and similar initial translation speeds in E. coli were used to confirm changes in olfactory receptor expression due to the AT10 tag. At this time, OR2B6, OR4F16, OR2W1, OR13C4, OR1L1, OR13D1, OR6F1, and OR6B2 were used as olfactory receptors, and the cell-free protein synthesis method was performed as described above. The results with and without the AT10 tag were compared to Table 6 and Figure 6. It is shown in 11.
(codon adaptation indes)CAI
(codon adaptation index)
(태그 없음)translation initiation rate (TIR)
(no tag)
(TA10 태그)TIR
(TA10 tag)
도 11 및 표 6에 나타난 바와 같이, AT10 태그가 없는 경우, 코돈 사용 빈도와 단백질 발현량이 유의적인 상관관계를 나타내지 않았다. 한편, AT10 태그를 사용한 경우에는 코돈 사용 빈도가 달라도 번역 초기속도가 모든 후각 수용체에서 현저히 증가하였을 뿐 아니라, 발현 편차 또한 유의적으로 감소하였다.As shown in Figure 11 and Table 6, in the absence of the AT10 tag, there was no significant correlation between codon usage frequency and protein expression level. Meanwhile, when the AT10 tag was used, not only did the initial translation speed significantly increase in all olfactory receptors, even if the codon usage frequency was different, but the expression deviation also significantly decreased.
실시예 8. AT10 태그에 의한 후각 수용체 발현 증가 확인-(3)Example 8. Confirmation of increased olfactory receptor expression by AT10 tag-(3)
상기에서 AT10 태그에 의한 발현 증가가 확인된 후각 수용체 중, 대장균에서 코돈 사용 빈도가 동일하면서 번역 초기속도가 다른 후각 수용체를 사용하여 AT10 태그에 의한 후각 수용체 발현 변화를 확인하였다. 이때, 후각 수용체로는 OR1D2, OR3A3, OR10K1, OR1N1, OR13D1, OR9A2, OR52E5, OR1L4 및 OR2J2를 사용하여 웨스턴 블롯을 수행하였으며 AT10 태그 유무에 따른 결과를 비교하여 표 7 및 도 12에 나타내었다.Among the olfactory receptors whose expression was confirmed to be increased by the AT10 tag above, olfactory receptors with the same codon usage frequency but different initial translation speeds in E. coli were used to confirm changes in olfactory receptor expression due to the AT10 tag. At this time, Western blot was performed using OR1D2, OR3A3, OR10K1, OR1N1, OR13D1, OR9A2, OR52E5, OR1L4, and OR2J2 as olfactory receptors, and the results with and without the AT10 tag were compared and shown in Table 7 and Figure 12.
도 12 및 표 7에 나타난 바와 같이, AT10 태그가 없는 경우, 번역 초기속도가 빠를수록 단백질 발현량이 유의적으로 증가하여 유의적인 상관관계를 나타내는 것을 알 수 있었다. 한편, AT10 태그를 사용한 경우에는 번역 초기속도가 다른 모든 후각 수용체에서 발현이 현저히 증가하였을 뿐 아니라, 발현 편차 또한 유의적으로 감소하였다.As shown in Figure 12 and Table 7, in the absence of the AT10 tag, the faster the initial translation speed, the higher the protein expression level, indicating a significant correlation. Meanwhile, when the AT10 tag was used, not only did the expression significantly increase in all olfactory receptors with different initial translation speeds, but the expression deviation also significantly decreased.
따라서, 상기 내용에 기초하였을 때, 박테리아 내에서 재조합 단백질의 발현은 코돈 사용 빈도와도 관계가 있으나, 이보다는 번역 초기속도가 더욱 유의적인 관계를 나타냄을 알 수 있었다.Therefore, based on the above, it was found that although the expression of recombinant proteins in bacteria is related to the frequency of codon use, the initial speed of translation shows a more significant relationship.
실시예 9. AT10 태그에 의한 GPCR 단백질의 발현 증가 확인Example 9. Confirmation of increased expression of GPCR protein by AT10 tag
후각 수용체를 포함하는 GPCR(G protein-coupled receptor) 또한 TA10 태그에 의해 발현 변화가 있는지 여부를 확인하였다. 실험은 GPCR인 CCR3(C-C motif chemokine receptor 3), CCR4(C-C motif chemokine receptor 4), CXCR2(C-X-C motif chemokine receptor 2), ADORA2A(adenosine A2a receptor), EDNRB(endothelin receptor type B), APLNR(apelin receptor) 또는 PTH1R(parathyroid hormone 1 receptor)을 암호화하는 유전자와 하기 표 8에 기재된 프라이머를 사용한 것으로 제외하고는 상기 서술한 바와 같은 방법으로 수행되었다. 그 결과, 후각 수용체의 발현정도를 확인한 웨스턴 블롯 결과를 도 13에 나타내었다.We also confirmed whether there was a change in expression of G protein-coupled receptor (GPCR), which includes olfactory receptors, due to the TA10 tag. The experiment was conducted on GPCRs: CCR3 (C-C motif chemokine receptor 3), CCR4 (C-C motif chemokine receptor 4), CXCR2 (C-X-C motif chemokine receptor 2), ADORA2A (adenosine A2a receptor), EDNRB (endothelin receptor type B), and APLNR (apelin receptor). ) or the gene encoding PTH1R (parathyroid hormone 1 receptor) and the primers listed in Table 8 below were used. As a result, the Western blot results confirming the expression level of olfactory receptors are shown in Figure 13.
도 13에 나타난 바와 같이, 후각 수용체 이외에도 다른 GPCR 또한 TA10 태그에 의해 발현이 유의적으로 증가하였다.As shown in Figure 13, in addition to the olfactory receptor, the expression of other GPCRs was also significantly increased by the TA10 tag.
실시예 10. 대장균에서 AT10 태그에 의한 GPCR 단백질의 발현 증가 확인Example 10. Confirmation of increased expression of GPCR protein by AT10 tag in E. coli
GPCR 단백질인 OR2B6, OR13C4, CCR3 또는 PTH1R을 암호화하는 유전자가 대장균에서 TA10 태그에 의해 발현 변화를 나타내는지 다음과 같이 확인하였다.It was confirmed as follows whether genes encoding GPCR proteins OR2B6, OR13C4, CCR3, or PTH1R showed expression changes due to the TA10 tag in E. coli.
먼저, OR2B6, OR13C4, CCR3 또는 PTH1R을 암호화하는 유전자를 상기 서술한 바와 같이 AT10이 포함되도록 발현벡터에 클로닝하고, 클로닝된 발현벡터를 통상적인 방법으로 BL21 세포주에 형질전환시켰다. 발현벡터가 형질전환된 벡터를 OD600에서 0.7에 도달할 때까지 배양하고, 1 mM의 IPTG를 처리하였다. 이를 4시간 더 배양하고 배양액을 원심분리하여 세포를 회수하였다. 초음파를 이용하여 통상적인 조건으로 세포를 파쇄시켜 세포 파쇄물을 수득하고, 이를 다시 원심분리하여 펠렛을 취하였다. 상기 펠렛에 가용화 완충액(20 mM Tris-HCl(pH 8.0), 20 mM SDS, 100 mM DTT, 1 mM EDTA)을 첨가하고 30℃에서 하룻밤 동안 반응시켰다. 가용화된 샘플을 사용하여 상기 서술한 바와 같이 웨스턴 블롯을 수행하고, 그 결과를 도 14에 나타내었다.First, genes encoding OR2B6, OR13C4, CCR3, or PTH1R were cloned into an expression vector to include AT10 as described above, and the cloned expression vector was transformed into the BL21 cell line by a conventional method. The vector transformed with the expression vector was cultured at OD 600 until it reached 0.7, and then treated with 1 mM IPTG. This was cultured for an additional 4 hours, and the culture medium was centrifuged to recover the cells. Cells were disrupted under normal conditions using ultrasound to obtain cell lysate, which was centrifuged again to obtain a pellet. Solubilization buffer (20mM Tris-HCl (pH 8.0), 20mM SDS, 100mM DTT, 1mM EDTA) was added to the pellet and reacted at 30°C overnight. Western blot was performed as described above using the solubilized samples, and the results are shown in Figure 14.
도 14에 나타난 바와 같이, 모든 GPCR 단백질이 대장균에서 AT10 태그에 의해 유의적으로 발현이 증가하였다.As shown in Figure 14, the expression of all GPCR proteins was significantly increased by the AT10 tag in E. coli.
실시예 11. AT10 태그된 GPCR 단백질의 활성 확인Example 11. Confirmation of activity of AT10 tagged GPCR protein
AT10으로 태그된 GPCR 단백질의 활성변화를 트립토판 형광 소광 분석법으로 확인하였다.Changes in the activity of the GPCR protein tagged with AT10 were confirmed using tryptophan fluorescence quenching analysis.
먼저, OR2W1 또는 PTH1R을 암호화하는 유전자를 상기 서술한 바와 같이 AT10이 포함되도록 발현벡터에 클로닝하고, 상기 서술한 바와 같이 대장균에 형질전환하여 세포 파쇄물을 수득하였다. 수득된 세포 파쇄물을 원심분리하여 봉입체(inclusion body) 형태의 GPCR 단백질을 얻고 가용화 완충액을 첨가하고 30℃에서 하룻밤동안 반응시켜 단백질을 가용화시켰다. 가용화된 GPCR 단백질을 결합 완충액(20 mM Tris-HCl(pH 8.0), 10 mM SDS)으로 투석하고, Ni-NTA 컬럼을 사용하여 통상적인 방법으로 정제하였다. 정제된 GPCR 단백질을 구조 재형성(refolding) 완충액(5 mM GSH, 1 mM GSSG, 6 mM n-도데실-β-D-말토피라노사이드(DDM), 6 mM 6-사이클로헥실헥실-β-D-말토사이드(Cymal6), 6 mM 메틸-β-사이클로덱스트린)을 첨가하여 통상적인 방법으로 단백질 구조를 재형성시켰다. 구조 재형성된 GPCR 단백질을 사용하여 트립토판 형광 소광 분석법으로 GPCR의 활성을 확인하고, 그 결과를 도 15에 나타내었다.First, the gene encoding OR2W1 or PTH1R was cloned into an expression vector containing AT10 as described above, and transformed into E. coli to obtain a cell lysate as described above. The obtained cell lysate was centrifuged to obtain the GPCR protein in the form of an inclusion body, and solubilization buffer was added and reacted at 30°C overnight to solubilize the protein. The solubilized GPCR protein was dialyzed against binding buffer (20mM Tris-HCl (pH 8.0), 10mM SDS) and purified by a conventional method using a Ni-NTA column. Purified GPCR proteins were incubated in refolding buffer (5 mM GSH, 1 mM GSSG, 6 mM n-dodecyl-β-D-maltopyranoside (DDM), 6 mM 6-cyclohexylhexyl-β- D-maltoside (Cymal6) and 6 mM methyl-β-cyclodextrin) were added to reform the protein structure in a conventional manner. The activity of the GPCR was confirmed using a tryptophan fluorescence quenching assay using the restructured GPCR protein, and the results are shown in Figure 15.
도 15에 나타난 바와 같이, AT10 태그가 GPCR 단백질의 활성에는 영향을 미치지 않음을 확인하였다.As shown in Figure 15, it was confirmed that the AT10 tag did not affect the activity of the GPCR protein.
Claims (13)
A tag for enhancing the expression of a membrane protein consisting of adenine (A) and thymine (T) and consisting of 3 to 99 nucleotides.
The tag for enhancing membrane protein expression according to claim 1, wherein the tag encodes lysine (K) or tyrosine (Y).
The tag for enhancing membrane protein expression according to claim 1, wherein KYY is sequentially added from the C-terminus of the first amino acid translated by the tag.
The tag for enhancing membrane protein expression according to claim 1, wherein the nucleotides are included in multiples of 3.
The tag for enhancing membrane protein expression according to claim 1, which consists of 3 to 66 nucleotides.
The tag for enhancing membrane protein expression according to claim 1, which consists of 3 to 33 nucleotides.
The method of claim 1, wherein the membrane protein is a G protein-coupled receptor (GPCR), a hormone receptor, a membrane transport protein, a cell adhesion molecule, or an enzyme. A tag for enhancing membrane protein expression, an enzyme-linked receptor.
The tag for enhancing membrane protein expression according to claim 7, wherein the GPCR is a GPCR of class A, class B, class C, class D, class E, or class F.
The tag for enhancing membrane protein expression according to claim 1, wherein the tag for enhancing membrane protein expression increases the initial expression rate of the protein.
An expression vector containing the tag for enhancing membrane protein expression of claim 1.
The expression vector according to claim 10, wherein the tag for enhancing membrane protein expression is inserted after the start codon of the membrane protein to be expressed.
A host cell transformed with the expression vector of claim 10.
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