KR20230066933A - Recombinant tyrosyl-tRNA synthetase and method for producing protein using the same - Google Patents
Recombinant tyrosyl-tRNA synthetase and method for producing protein using the same Download PDFInfo
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- KR20230066933A KR20230066933A KR1020210152468A KR20210152468A KR20230066933A KR 20230066933 A KR20230066933 A KR 20230066933A KR 1020210152468 A KR1020210152468 A KR 1020210152468A KR 20210152468 A KR20210152468 A KR 20210152468A KR 20230066933 A KR20230066933 A KR 20230066933A
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- South Korea
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- recombinant
- tyrosyl
- trna synthetase
- protein
- leu
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Abstract
Description
본 발명은 재조합 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase) 및 이를 이용한 단백질 생산 방법에 관한 것으로서, 더욱 상세하게는 단백질 내의 앰버 코돈(amber codon)위치에 4-아지도-L-페닐알라닌(4-azido-L-phenylalanine)을 결합시킬 수 있는 재조합 티로실-tRNA 합성효소를 설계하여, 합성된 유전자 DNA를 이용한 4-아지도-L-페닐알라닌이 포함된 단백질의 생산방법에 관한 것으로서, 더욱 상세하게는 기존에 사용된 미생물 유래 돌연변이 티로실-tRNA 합성효소를 대체할 수 있는 새로운 재조합 티로실-tRNA 합성효소를 설계하고, 설계된 새로운 재조합 티로실-tRNA 합성효소의 유전자를 합성함으로써 대장균에서 발현시키는 방법에 관한 것이다.The present invention relates to a recombinant tyrosyl-tRNA synthetase and a method for producing a protein using the same, and more particularly, to a protein containing 4-azido-L-phenylalanine (4-azido-L-phenylalanine) at an amber codon position in a protein. -azido-L-phenylalanine) by designing a recombinant tyrosyl-tRNA synthetase capable of binding 4-azido-L-phenylalanine) and producing a protein containing 4-azido-L-phenylalanine using the synthesized DNA. In particular, by designing a new recombinant tyrosyl-tRNA synthetase that can replace the previously used microbial-derived mutant tyrosyl-tRNA synthetase and synthesizing the gene of the designed new recombinant tyrosyl-tRNA synthetase, expression in E. coli It's about how.
또한, 새로운 재조합 티로실-tRNA 합성효소를 이용하여 앰버 코돈(amber codon)의 위치에 4-아지도-L-페닐알라닌이 결합되어 있는 재조합 단백질을 생산하는 방법에 관한 것이다.In addition, it relates to a method for producing a recombinant protein in which 4-azido-L-phenylalanine is linked to an amber codon position using a novel recombinant tyrosyl-tRNA synthetase.
단백질은 거의 모든 생물학적 물질에 관여하지만, 동일한 20개의 공통 아미노산에서 생합성되며 질소 염기, 카르복시산, 아미드, 알코올 및 티올과 같은 공통된 기능기만을 가지고 있다. 때문에, 단백질을 이루고 있는 아미노산에 비천연 기능기를 결합할 수 있다면 단백질의 기능기 치환이나 단백질 구조의 변화 등을 가능하게 하는 강력한 도구를 제공할 수 있다.Proteins are involved in almost all biological substances, but they are biosynthesized from the same 20 common amino acids and have only common functionalities such as nitrogenous bases, carboxylic acids, amides, alcohols and thiols. Therefore, if a non-natural functional group can be bound to an amino acid constituting a protein, it can provide a powerful tool that enables functional group substitution of a protein or change in protein structure.
자연계에 일반적으로 존재하는 20종의 아미노산이 아닌 새로운 생화학적 특성을 나타내는 비천연 아미노산을 단백질의 특정 위치에 삽입하는 기술은 물리적, 화학적 및 생물학적 특성이 변화된 새로운 단백질을 만드는 좋은 방법이 될 수 있을 뿐만 아니라 단백질의 기능연구에도 활용될 수 있다.The technology of inserting non-natural amino acids that exhibit new biochemical properties, rather than the 20 amino acids commonly found in nature, at specific positions in proteins can be a good way to create new proteins with altered physical, chemical, and biological properties. It can also be used for functional studies of proteins.
이에 따라, 최근 이와 같은 단백질을 이루고 있는 아미노산에 비천연 기능기를 결합시키기 위한 연구가 활발히 진행되었다. Chin 등은 일반적인 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase; TyrRS) 대신에 메타노코커스 자나쉬아이(Methanococcus jannaschii)에서 유래한 티로실-tRNA 합성효소 돌연변이체를 개발하여 특정 티로신(tyrosine) 부위에 선택적으로 4-아지도-L-페닐알라닌이 결합된 단백질을 생산하는 기술을 개발하였다.Accordingly, studies have recently been actively conducted to combine non-natural functional groups with amino acids constituting such proteins. Chin et al. developed a tyrosyl-tRNA synthetase mutant derived from Methanococcus jannaschii instead of the general tyrosyl-tRNA synthetase (TyrRS) to generate a specific tyrosine site. developed a technology for producing a protein to which 4-azido-L-phenylalanine is selectively bound.
이렇게 비천연 기능기가 포함된 비천연 아미노산을 포함하는 단백질은 기존의 천연 아미노산만을 포함하는 단백질에 비하여 선택적 화학반응을 통하여 단백질의 안정성을 높일 수 있었다. Lim 등은 이러한 비천연 아미노산이 포함된 단백질을 이용하여 체내에서 안전성이 증가된 유레이트 옥시다제(urate oxidase)를 생산할 수 있는 재조합 대장균을 개발하였다.Compared to proteins containing only existing natural amino acids, proteins containing non-natural amino acids containing non-natural functional groups can increase protein stability through selective chemical reactions. Lim et al. developed a recombinant E. coli capable of producing urate oxidase with increased safety in the body using a protein containing such unnatural amino acids.
다양한 비천연 아미노산이 포함된 재조합 단백질의 생산을 위해서는 먼저 비천연 아미노산으로 치환하고자 하는 위치에서 앰버 코돈으로 유전자의 염기서열을 변화시켜야 하고, 앰버 서프레션이 가능하면서 비천연 아미노산을 결합할 수 있는 특별한 돌연변이 티로실-tRNA 합성효소가 필요하다.In order to produce a recombinant protein containing various unnatural amino acids, the nucleotide sequence of the gene must first be changed to an amber codon at the position to be substituted with a non-natural amino acid, and a special protein that can bind non-natural amino acids while suppressing amber is required. A mutant tyrosyl-tRNA synthetase is required.
현재 사용되고 있는 대표적인 돌연변이 티로실-tRNA 합성효소는 메타노코커스 자나쉬아이에서 유래한 돌연변이 티로실-tRNA 합성효소이다. 현재 다양한 돌연변이 티로실-tRNA 합성효소들이 보고되었다. 하지만, 기존 돌연변이 티로실-tRNA 합성효소의 개량과 더욱 다양한 비천연 아미노산을 단백질에 첨가하기 위해서는 기존에 보고된 돌연변이 티로실-tRNA 합성효소들의 단백질 구조 분석으로부터 새로운 재조합 티로실-tRNA 합성효소를 설계할 필요가 있다.A representative mutant tyrosyl-tRNA synthetase currently used is Methanococcus janashii. It is a mutant tyrosyl-tRNA synthetase derived from Currently, various mutant tyrosyl-tRNA synthetases have been reported. However, in order to improve existing mutant tyrosyl-tRNA synthetases and to add more diverse unnatural amino acids to proteins, design new recombinant tyrosyl-tRNA synthetases from protein structure analysis of previously reported mutant tyrosyl-tRNA synthetases. Needs to be.
이에 본 발명자들은 목적단백질 내의 앰버 코돈(amber codon)위치에 4-아지도-L-페닐알라닌(4-azido-L-phenylalanine)를 결합시킬 수 있는 재조합 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase)를, 기존의 돌연변이 유도 방법이 아닌 화학적인 DNA 합성 방법으로 설계하였고, 이를 이용하여 앰버 코돈의 위치에 4-아지도-L-페닐알라닌이 결합되어 있는 재조합 단백질을 생산할 수 있음을 확인하였다.Accordingly, the present inventors developed a recombinant tyrosyl-tRNA synthetase capable of binding 4-azido-L-phenylalanine to the amber codon position in the target protein. was designed by a chemical DNA synthesis method rather than a conventional mutagenesis method, and it was confirmed that a recombinant protein in which 4-azido-L-phenylalanine was bound at the position of an amber codon could be produced using this method.
이에, 본 발명의 목적은 서열번호 1 또는 3으로 표시되는 아미노산 서열을 포함하는 재조합 티로실-tRNA 합성효소를 제공하는 것이다.Accordingly, an object of the present invention is to provide a recombinant tyrosyl-tRNA synthetase comprising the amino acid sequence represented by SEQ ID NO: 1 or 3.
본 발명의 다른 목적은 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터를 제공하는 것이다.Another object of the present invention is to provide a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase.
본 발명의 또 다른 목적은 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 형질전환된 미생물을 제공하는 것이다.Another object of the present invention is to provide a microorganism transformed with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase.
본 발명의 또 다른 목적은 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 미생물을 형질전환하는 형질전환 단계를 포함하는 재조합 티로실-tRNA 합성효소 생산 미생물의 제조 방법을 제공하는 것이다.Another object of the present invention is a recombinant tyrosyl-tRNA synthesis comprising a transformation step of transforming a microorganism with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase. It is to provide a method for producing enzyme-producing microorganisms.
본 발명의 또 다른 목적은 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 형질전환된 미생물을 배양하는 배양 단계를 포함하는 단백질 생산 방법을 제공하는 것이다.Another object of the present invention is to provide a protein production method comprising a culturing step of culturing a microorganism transformed with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase. is to do
본 발명은 재조합 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase) 및 이를 이용한 단백질 생산 방법에 관한 것으로, 본 발명에 따른 재조합 티로실-tRNA 합성효소는 단백질 내의 앰버 코돈(amber codon)위치에 4-아지도-L-페닐알라닌(4-azido-L-phenylalanine)을 결합시킬 수 있다.The present invention relates to a recombinant tyrosyl-tRNA synthetase and a method for producing a protein using the same. The recombinant tyrosyl-tRNA synthetase according to the present invention contains 4- Azido-L-phenylalanine (4-azido-L-phenylalanine) can be bound.
본 발명자들은 기존에 보고된 티로실-tRNA 합성효소 돌연변이들의 단백질 정보로부터 새로운 재조합 티로실-tRNA 합성효소를 설계하여 CHI 및 JNCC로 각각 명명하고, 이로부터 앰버 코돈의 서프레션과 비천연 아미노산의 결합 가능성을 확인하였다.The present inventors designed a new recombinant tyrosyl-tRNA synthetase from protein information of previously reported tyrosyl-tRNA synthetase mutants and named them CHI and JNCC, respectively, from which amber codon suppression and unnatural amino acid binding possibility was confirmed.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 서열번호 1 또는 3으로 표시되는 아미노산 서열을 포함하는 재조합 티로실-tRNA 합성효소이다.One aspect of the present invention is a recombinant tyrosyl-tRNA synthetase comprising the amino acid sequence represented by SEQ ID NO: 1 or 3.
본 발명의 일 구현예에서, 상기 서열번호 1로 표시되는 아미노산 서열을 포함하는 재조합 티로실-tRNA 합성효소는 CHI, 서열번호 3으로 표시되는 아미노산 서열을 포함하는 재조합 티로실-tRNA 합성효소는 JNCC로 명명하였다.In one embodiment of the present invention, the recombinant tyrosyl-tRNA synthetase comprising the amino acid sequence represented by SEQ ID NO: 1 is CHI, and the recombinant tyrosyl-tRNA synthetase comprising the amino acid sequence represented by SEQ ID NO: 3 is JNCC named as
본 발명의 다른 양태는 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터이다.Another aspect of the present invention is a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase.
본 명세서상의 용어 “벡터(vector)”는 숙주세포에서 목적 유전자를 발현시키기 위한 수단을 의미한다. 예를 들어, 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노연관 바이러스 벡터와 같은 바이러스 벡터를 포함한다. 재조합 벡터로 사용될 수 있는 벡터는 당업계에서 종종 사용되는 플라스미드(예를 들면, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예를 들면, λgt4λB, λ-Charon, λΔz1 및 M13 등) 또는 바이러스(예를 들면, SV40 등)를 조작하여 제작될 수 있으나 이에 제한되지 않는다.The term "vector" used herein refers to a means for expressing a gene of interest in a host cell. Examples include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as recombinant vectors include plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, It may be produced by manipulating pGEX series, pET series and pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.), but is not limited thereto.
상기 재조합 벡터는, 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 상기 발현용 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 단백질을 발현하는 데 사용되는 통상의 것을 사용할 수 있다. 상기 재조합 벡터는 당업계에 공지된 다양한 방법을 통해 구축될 수 있다.The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. As the expression vector, conventional ones used in the art to express foreign proteins in plants, animals, or microorganisms may be used. The recombinant vector may be constructed through various methods known in the art.
상기 재조합 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다. 예를 들어, 사용되는 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예를 들어, pLλ 프로모터, CMV 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 진핵 세포를 숙주로 하는 경우에는, 벡터에 포함되는 진핵 세포에서 작동하는 복제원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 및 BBV 복제원점 등을 포함하나, 이에 한정되는 것은 아니다. 또한, 포유동물 세포의 게놈으로부터 유래된 프로모터(예를 들어, 메탈로티오닌 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예를 들어, 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토메갈로바이러스 프로모터 및 HSV의 tk 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.The recombinant vector may be constructed using a prokaryotic or eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (eg, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. In the case of using a eukaryotic cell as a host, the origin of replication operating in the eukaryotic cell included in the vector includes the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication and the BBV origin of replication, etc. It is not limited. In addition, promoters derived from the genome of mammalian cells (eg, metallotionine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.
본 발명의 일 예에서, 재조합 벡터를 숙주세포에 삽입함으로써 형질전환체를 만들 수 있으며, 상기 형질전환체는 상기 재조합 벡터를 적절한 숙주세포에 도입시킴으로써 얻어진 것일 수 있다.In one example of the present invention, a transformant may be produced by inserting a recombinant vector into a host cell, and the transformant may be obtained by introducing the recombinant vector into an appropriate host cell.
상기 숙주세포는 상기 발현벡터를 안정되면서 연속적으로 클로닝 또는 발현시킬 수 있는 세포로서 당업계에 공지된 어떠한 숙주세포도 이용할 수 있다.As the host cell, any host cell known in the art may be used as a cell capable of stably and continuously cloning or expressing the expression vector.
본 발명에서 사용된 숙주세포로는 대장균, 효모, 동물세포, 식물세포, 또는 곤충세포 등을 포함할 수 있으며, 원핵세포로는, 예를 들어, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있으며, 진핵 세포에 형질 전환시키는 경우에는 숙주세포로서, 효모(Saccharomyce cerevisiae), 곤충 세포, 식물 세포 및 동물 세포, 예를 들어, Sp2/0, CHO(Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK 세포주 등이 이용될 수 있으나, 이에 제한되는 것은 아니다.Host cells used in the present invention may include Escherichia coli, yeast, animal cells, plant cells, or insect cells, and prokaryotic cells include, for example, E. coli JM109, E. coli BL21, E. strains of the genus Bacillus, such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcessons and There are enterobacteriaceae strains such as various Pseudomonas species, and when transformed into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. may be used, but are not limited thereto.
상기 폴리뉴클레오타이드 또는 이를 포함하는 재조합 벡터의 숙주세포 내로의 운반(도입)은, 당업계에 널리 알려진 운반 방법을 사용할 수 있다. 상기 운반 방법은 예를 들어, 숙주세포가 원핵 세포인 경우, CaCl2 방법 또는 전기 천공 방법 등을 사용할 수 있고, 숙주세포가 진핵 세포인 경우에는, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀매개 형질감염법 및 유전자 밤바드먼트 등을 사용할 수 있으나, 이에 한정하지는 않는다.Transport (introduction) of the polynucleotide or a recombinant vector containing the polynucleotide into a host cell may use a transport method widely known in the art. As the delivery method, for example, when the host cell is a prokaryotic cell, a CaCl 2 method or an electroporation method may be used, and when the host cell is a eukaryotic cell, a microinjection method, a calcium phosphate precipitation method, an electroporation method, Liposome-mediated transfection and gene bombardment may be used, but are not limited thereto.
상기 형질 전환된 숙주세포를 선별하는 방법은 선택 표지에 의해 발현되는 표현형을 이용하여, 당업계에 널리 알려진 방법에 따라 용이하게 실시할 수 있다. 예를 들어, 상기 선택 표지가 특정 항생제 내성 유전자인 경우에는, 상기 항생제가 함유된 배지에서 형질전환체를 배양함으로써 형질전환체를 용이하게 선별할 수 있다.The method for selecting the transformed host cell can be easily performed according to a method widely known in the art using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.
본 발명의 또 다른 양태는 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 형질전환된 미생물이다.Another aspect of the present invention is a microorganism transformed with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase.
본 발명에 있어서 미생물은 대장균(Escherichia coli; E. coli)인 것일 수 있고, 상기 서열번호 2 또는 4로 표시되는 핵산 서열은 대장균에서 발현시키기 위하여 코돈 최적화가 진행된 것일 수 있다.In the present invention, the microorganism may be Escherichia coli ( E. coli ), and the nucleic acid sequence represented by SEQ ID NO: 2 or 4 may be codon-optimized for expression in E. coli.
상기 미생물은 넌센스 코돈이 포함된 목적단백질을 코딩하는 핵산 서열로 추가적으로 형질전환된 것일 수 있다.The microorganism may be additionally transformed with a nucleic acid sequence encoding a target protein containing a nonsense codon.
본 명세서상의 용어 “목적단백질”은 단백질 생산 방법의 실시에 따라 획득하고자 하는 단백질을 의미한다.The term "target protein" used herein refers to a protein to be obtained by performing a protein production method.
상기 목적단백질은 형광단백질(superfolder green fluorescent protein, sfGFP) 또는 이의 재조합체인 것일 수 있으나, 이에 한정되는 것은 아니다.The target protein may be a fluorescent protein (superfolder green fluorescent protein, sfGFP) or a recombinant thereof, but is not limited thereto.
상기 넌센스 코돈은 앰버 코돈(amber codon), 오커 코돈(ochre codon), 오팔 코돈(opal codon), 특이 코돈(unique codon), 희귀 코돈(rare codon) 및 4-염기 코돈으로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, TAG 앰버 코돈인 것일 수 있으나, 이에 한정되는 것은 아니다.The nonsense codon is selected from the group consisting of an amber codon, an ocher codon, an opal codon, a unique codon, a rare codon and a 4-base codon 1 It may be more than one species, for example, it may be a TAG amber codon, but is not limited thereto.
본 발명의 일 구현예에서, 목적단백질을 코딩하는 핵산 서열에 종결코돈으로 인식되는 넌센스 코돈, 예를 들어 앰버 코돈이 포함되어 있는 경우 단백질의 발현이 되지 않을 수 있다. 이 때 앰버 코돈에 페닐알라닌을 결합시키면 앰버 코돈이 억제되어 전사가 종결되지 않으므로 목적단백질이 완전히 합성되어 발현될 수 있다.In one embodiment of the present invention, if a nonsense codon recognized as a stop codon, for example, an amber codon, is included in the nucleic acid sequence encoding the target protein, the protein may not be expressed. At this time, when phenylalanine is bound to the amber codon, the amber codon is suppressed and transcription is not terminated, so the target protein can be completely synthesized and expressed.
상기 형질전환은 재조합 티로실-tRNA 합성효소를 코딩하는 핵산 서열 및 목적단백질을 코딩하는 핵산 서열을 동일한 하나의 재조합 벡터에 포함하여 수행되는 것일 수 있고, 또는 각각의 독립적인 재조합 벡터에 포함하여 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다.The transformation may be carried out by including the nucleic acid sequence encoding the recombinant tyrosyl-tRNA synthetase and the nucleic acid sequence encoding the target protein in the same recombinant vector, or by including them in each independent recombinant vector. It may be, but is not limited thereto.
본 발명의 또 다른 양태는 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 미생물을 형질전환하는 형질전환 단계를 포함하는 재조합 티로실-tRNA 합성효소 생산 미생물의 제조 방법이다.Another aspect of the present invention is a recombinant tyrosyl-tRNA synthesis comprising a transformation step of transforming a microorganism with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase It is a method for producing enzyme-producing microorganisms.
본 발명에 있어서 미생물은 대장균인 것일 수 있고, 상기 서열번호 2 또는 4으로 표시되는 핵산 서열은 대장균에서 발현시키기 위하여 코돈 최적화가 진행된 것일 수 있다.In the present invention, the microorganism may be Escherichia coli, and the nucleic acid sequence represented by SEQ ID NO: 2 or 4 may be codon-optimized for expression in Escherichia coli.
상기 미생물은 넌센스 코돈이 포함된 목적단백질을 코딩하는 핵산 서열로 추가적으로 형질전환된 것일 수 있다.The microorganism may be additionally transformed with a nucleic acid sequence encoding a target protein containing a nonsense codon.
상기 목적단백질은 형광단백질 또는 이의 재조합체인 것일 수 있으나, 이에 한정되는 것은 아니다.The target protein may be a fluorescent protein or a recombinant thereof, but is not limited thereto.
상기 넌센스 코돈은 앰버 코돈, 오커 코돈, 오팔 코돈, 특이 코돈, 희귀 코돈 및 4-염기 코돈으로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, TAG 앰버 코돈인 것일 수 있으나, 이에 한정되는 것은 아니다.The nonsense codon may be at least one selected from the group consisting of an amber codon, an ocher codon, an opal codon, a specific codon, a rare codon, and a 4-base codon, and may be, for example, a TAG amber codon, but is limited thereto it is not going to be
상기 형질전환 단계는 재조합 티로실-tRNA 합성효소를 코딩하는 핵산 서열 및 목적단백질을 코딩하는 핵산 서열을 동일한 하나의 재조합 벡터에 포함하여 수행되는 것일 수 있고, 또는 각각의 독립적인 재조합 벡터에 포함하여 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다.The transformation step may be carried out by including the nucleic acid sequence encoding the recombinant tyrosyl-tRNA synthetase and the nucleic acid sequence encoding the target protein in the same recombinant vector, or by including them in each independent recombinant vector. It may be performed, but is not limited thereto.
본 발명의 또 다른 양태는 재조합 티로실-tRNA 합성효소를 코딩하는 서열번호 2 또는 4로 표시되는 핵산 서열을 포함하는 재조합 벡터로 형질전환된 미생물을 배양하는 배양 단계를 포함하는 단백질 생산 방법이다.Another aspect of the present invention is a protein production method comprising a culturing step of culturing a microorganism transformed with a recombinant vector comprising a nucleic acid sequence represented by SEQ ID NO: 2 or 4 encoding a recombinant tyrosyl-tRNA synthetase.
본 발명에 있어서 미생물은 대장균인 것일 수 있고, 상기 서열번호 2 또는 4로 표시되는 핵산 서열은 대장균에서 발현시키기 위하여 코돈 최적화가 진행된 것일 수 있다.In the present invention, the microorganism may be Escherichia coli, and the nucleic acid sequence represented by SEQ ID NO: 2 or 4 may be codon-optimized for expression in Escherichia coli.
상기 미생물은 넌센스 코돈이 포함된 목적단백질을 코딩하는 핵산 서열로 추가적으로 형질전환된 것일 수 있다.The microorganism may be additionally transformed with a nucleic acid sequence encoding a target protein containing a nonsense codon.
상기 목적단백질은 형광단백질 또는 이의 재조합체인 것일 수 있으나, 이에 한정되는 것은 아니다.The target protein may be a fluorescent protein or a recombinant thereof, but is not limited thereto.
상기 넌센스 코돈은 앰버 코돈, 오커 코돈, 오팔 코돈, 특이 코돈, 희귀 코돈 및 4-염기 코돈으로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, TAG 앰버 코돈인 것일 수 있으나, 이에 한정되는 것은 아니다.The nonsense codon may be at least one selected from the group consisting of an amber codon, an ocher codon, an opal codon, a specific codon, a rare codon, and a 4-base codon, and may be, for example, a TAG amber codon, but is limited thereto it is not going to be
본 발명의 일 구현예에서, 목적단백질을 코딩하는 핵산 서열에 종결코돈으로 인식되는 넌센스 코돈, 예를 들어 앰버 코돈이 포함되어 있는 경우 단백질의 발현이 되지 않을 수 있다. 이 때 앰버 코돈에 페닐알라닌을 결합시키면 앰버 코돈이 억제되어 전사가 종결되지 않으므로 목적단백질이 완전히 합성되어 발현될 수 있다.In one embodiment of the present invention, if a nonsense codon recognized as a stop codon, for example, an amber codon, is included in the nucleic acid sequence encoding the target protein, the protein may not be expressed. At this time, when phenylalanine is bound to the amber codon, the amber codon is suppressed and transcription is not terminated, so the target protein can be completely synthesized and expressed.
상기 형질전환은 재조합 티로실-tRNA 합성효소를 코딩하는 핵산 서열 및 목적단백질을 코딩하는 핵산 서열을 동일한 하나의 재조합 벡터에 포함하여 수행되는 것일 수 있고, 또는 각각의 독립적인 재조합 벡터에 포함하여 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다.The transformation may be carried out by including the nucleic acid sequence encoding the recombinant tyrosyl-tRNA synthetase and the nucleic acid sequence encoding the target protein in the same recombinant vector, or by including them in each independent recombinant vector. It may be, but is not limited thereto.
본 발명에 있어서 배양 단계는 미생물에서 넌센스 코돈이 포함된 목적단백질을 코딩하는 유전자가 발현되는 것일 수 있다. 상기 목적단백질은 유전자로부터 발현이 이루어지는 과정에서 재조합 티로실-tRNA 합성효소에 의한 반응에 따라, 목적단백질을 이루는 아미노산에 특정한 기능기가 결합된 상태로 생산되는 것일 수 있다.In the present invention, the culturing step may be the expression of a gene encoding a target protein containing a nonsense codon in a microorganism. The target protein may be produced in a state in which a specific functional group is bound to an amino acid constituting the target protein according to a reaction by a recombinant tyrosyl-tRNA synthetase in the process of expression from a gene.
본 발명에 있어서 배양 단계는 천연 또는 비천연 아미노산을 포함하는 배지에서 수행되는 것일 수 있고, 예를 들어, 상기 비천연 아미노산은 4-아지도-L-페닐알라닌인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step may be performed in a medium containing a natural or non-natural amino acid, for example, the non-natural amino acid may be 4-azido-L-phenylalanine, but is not limited thereto. .
본 발명은 기존의 미생물에서 유래한 돌연변이 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase)와는 달리, 보고된 돌연변이 티로실-tRNA 합성효소들의 단백질 구조를 분석하여 설계된 새로운 아미노산 서열을 갖는 재조합 티로실-tRNA 합성효소 CHI 및 JNCC에 관한 것으로서, 설계된 재조합 티로실-tRNA 합성효소 CHI 및 JNCC를 대장균에서 효율적으로 발현시키기 위하여 대장균의 코돈 사용에 최적화된 DNA 서열로 합성한 유전자를 이용한다.Unlike mutant tyrosyl-tRNA synthetases derived from conventional microorganisms, the present invention analyzes the protein structures of reported mutant tyrosyl-tRNA synthetases and recombinant tyrosyl-tRNA synthetases having a new amino acid sequence designed. Regarding the tRNA synthetases CHI and JNCC, in order to efficiently express the designed recombinant tyrosyl-tRNA synthetases CHI and JNCC in E. coli, genes synthesized with DNA sequences optimized for use of codons in E. coli are used.
또한, 본 발명은 이를 이용하여 단백질의 특이 위치에 4-아지도-L-페닐알라닌(4-azido-L-phenylalanine)이 결합된 재조합 단백질을 대장균에서 발현하는 방법에 관한 것이다. 본 발명에서 얻어진 재조합 티로실-tRNA 합성효소 CHI 및 JNCC를 향후 다양한 비천연 아미노산의 결합과 활성 향상을 위한 개량 등에 활용할 수 있다.In addition, the present invention relates to a method for expressing in E. coli a recombinant protein to which 4-azido-L-phenylalanine is bound at a specific position of the protein using the same. The recombinant tyrosyl-tRNA synthetases CHI and JNCC obtained in the present invention can be used in the future to improve the binding and activity of various non-natural amino acids.
도 1은 본 발명의 일 실시예에 따라 재조합 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase) CHI가 형질전환된 대장균(Escherichia coli; E. coli)에서의 형광단백질 발현 결과 사진이다.
도 2는 본 발명의 일 실시예에 따라 재조합 티로실-tRNA 합성효소 JNCC가 형질전환된 대장균에서의 형광단백질 발현 결과 사진이다.1 is a photograph of a fluorescent protein expression result in Escherichia coli ( E. coli ) transformed with a recombinant tyrosyl-tRNA synthetase CHI according to an embodiment of the present invention.
Figure 2 is a photograph of the result of fluorescent protein expression in E. coli transformed with recombinant tyrosyl-tRNA synthetase JNCC according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피)%, 그리고 액체/액체는 (부피/부피)%이다.Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and liquid/liquid is (volume/volume) %.
실시예 1: 재조합 티로실-tRNA 합성효소 설계Example 1: Design of recombinant tyrosyl-tRNA synthetase
본 발명자는 기존에 사용되고 있는 메타노코커스 자나쉬아이(Methanococcus jannaschii)에서 유래한 돌연변이 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase; TyrRS)의 단백질 구조를 분석하였다. 단백질 구조 분석 결과로부터 앰버 서프레션 활성과 비천연 아미노산 4-아지도-L-페닐알라닌(4-azido-L-phenylalanine)의 결합능을 가질 수 있는 새로운 재조합 티로실-tRNA 합성효소의 후보군을 설계하였고, 그들 중에서 가장 가능성이 높은 2개의 재조합 티로실-tRNA 합성효소인 CHI 및 JNCC를 선정하였다.The present inventors analyzed the protein structure of a mutant tyrosyl-tRNA synthetase (TyrRS) derived from Methanococcus jannaschii, which is previously used. From the results of protein structure analysis, a candidate group of new recombinant tyrosyl-tRNA synthetase capable of binding amber suppression activity and non-natural amino acid 4-azido-L-phenylalanine was designed. Among them, the two most likely recombinant tyrosyl-tRNA synthetases, CHI and JNCC, were selected.
선정된 CHI 및 JNCC의 아미노산 서열은 각각 서열변호 1 및 3로 나타내었다. 설계된 재조합 재조합 티로실-tRNA 합성효소 CHI 및 JNCC의 아미노산 서열로부터 대장균에 발현이 용이한 코돈을 갖는 DNA 염기서열은 각각 서열번호 2 및 4로 나타내었다. 이렇게 설계된 재조합 티로실-tRNA 합성효소 CHI 및 JNCC의 염기서열은 합성서비스 업체(코스모진텍, 서울)를 통하여 화학적 합성방법에 의하여 합성되었다. 합성된 유전자들은 아래 방법으로 대장균에 클로닝되었다.The selected amino acid sequences of CHI and JNCC are shown as SEQ ID NOs. 1 and 3, respectively. From the amino acid sequences of the designed recombinant tyrosyl-tRNA synthetases CHI and JNCC, DNA sequences having codons that are easily expressed in E. coli are shown in SEQ ID NOs: 2 and 4, respectively. The base sequences of the recombinant tyrosyl-tRNA synthetases CHI and JNCC thus designed were synthesized by chemical synthesis through a synthesis service company (Cosmogenetech, Seoul). The synthesized genes were cloned into E. coli by the following method.
먼저 합성된 재조합 티로실-tRNA 합성효소 CHI와 JNCC의 염기서열을 주형으로 사용하여, 적절한 농도로 희석한 후 키트를 이용하여 PCR을 진행하였다. 사용된 프라이머의 염기서열은 하기 표 2와 같다.First, using the nucleotide sequences of the synthesized recombinant tyrosyl-tRNA synthetase CHI and JNCC as templates, diluted to appropriate concentrations, PCR was performed using a kit. Base sequences of the primers used are shown in Table 2 below.
얻어진 PCR 결과물은 제한효소 BglII와 PstI으로 절단하고, 같은 제한효소로 절단된 pEVOL-pAzF(Plasmid ID: 31186, Addgene(Cambridge, MA))에 라이게이즈(ligase)를 이용하여 재조합 벡터 pEVOL-CHI와 pEVOL-JNCC를 각각 제작하였다. The obtained PCR product was digested with restriction enzymes Bgl II and Pst I, and pEVOL-pAzF (Plasmid ID: 31186, Addgene (Cambridge, MA)) digested with the same restriction enzymes was ligated to recombinant vector pEVOL. -CHI and pEVOL-JNCC were prepared respectively.
실시예 2: 설계된 재조합 티로실-tRNA 합성효소의 앰버 서프레션 활성과 4-아지도-L-페닐알라닌 결합성 확인Example 2: Confirmation of amber suppression activity and 4-azido-L-phenylalanine binding of the designed recombinant tyrosyl-tRNA synthetase
이러한 방법으로 얻어진 재조합 티로실-tRNA 합성효소 CHI 및 JNCC의 앰버 서프레션 활성과 4-아지도-L-페닐알라닌의 선택성은 앰버 코돈(amber codon)이 있는 형광단백질(superfolder green fluorescent protein, sfGFP)의 발현을 확인함으로써 이루어졌다.The amber suppression activity of the recombinant tyrosyl-tRNA synthetases CHI and JNCC obtained in this way and the selectivity of 4-azido-L-phenylalanine were compared to superfolder green fluorescent protein (sfGFP) with an amber codon. This was done by checking the expression.
앰버 코돈이 있는 형광단백질은 원래의 형광단백질의 204번 아미노산 자리에 앰버 코돈을 치환하여 제작하였다. 앰버 코돈이 있는 형광단백질 유전자는 pSEVA631(Silva-Rocha R. et al. (2013) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41(Database issue):D666-75.)의 EcoRI과 KpnI 자리에 삽입하여 발현 벡터 pSEVA631-sfGFP를 제작하였다.A fluorescent protein with an amber codon was prepared by substituting an amber codon at amino acid position 204 of the original fluorescent protein. The fluorescent protein gene with an amber codon is pSEVA631 (Silva-Rocha R. et al. (2013) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res . 41 (Database issue): D666-75.) was inserted into the Eco RI and Kpn I sites to construct the expression vector pSEVA631-sfGFP.
대장균 DH10B(New England Biolabs, Ipswich, MA, USA) 컴피턴트 셀(competent cell)에 먼저 앰버 코돈이 있는 형광단백질 유전자를 포함하는 pSEVA631-sfGFP를 형질전환하여 젠타마이신(gentamycin)이 함유된 배지에서 형질전환체를 얻었다.Escherichia coli DH10B (New England Biolabs, Ipswich, MA, USA) competent cells were first transformed with pSEVA631-sfGFP containing a fluorescent protein gene with an amber codon in a medium containing gentamycin conversion was obtained.
얻어진 재조합 대장균을 컴피턴트 셀로 준비하고, 제작된 재조합 티로실-tRNA 합성효소 돌연변이체들을 포함하는 pEVOL-CHI와 pEVOL-JNCC 벡터를 각각 형질전환하여 두개의 벡터가 있는 재조합 대장균을 앰피실린(ampicillin) 및 젠타마이신 혼합 배지에서 선별하여 얻었다. 얻어진 형질전환체들을 4-아지도-L-페닐알라닌이 첨가된, 또는 첨가되지 않은 액체 배지에 접종하여 형광단백질의 발현 여부를 확인하였다.The resulting recombinant E. coli was prepared as a competent cell, and the pEVOL-CHI and pEVOL-JNCC vectors containing the prepared recombinant tyrosyl-tRNA synthetase mutants were respectively transformed to ampicillin the recombinant E. coli with the two vectors. and gentamicin mixed medium. The obtained transformants were inoculated in a liquid medium to which 4-azido-L-phenylalanine was added or not, and the expression of fluorescent protein was confirmed.
도 1에서 확인할 수 있듯이, 재조합 티로실-tRNA 합성효소 CHI를 갖고 있는 pEVOL-CHI와 pSEVA631-sfGFP가 클로닝된 재조합 대장균은 4-아지도-L-페닐알라닌이 첨가되지 않은 배지(상)에서는 형광을 보이지 않았지만, 4-아지도-L-페닐알라닌이 첨가된 배지(하)에서는 형광단백질의 발현이 확인되었다.As can be seen in Figure 1, the recombinant Escherichia coli cloned with pEVOL-CHI and pSEVA631-sfGFP having recombinant tyrosyl-tRNA synthetase CHI show fluorescence in a medium (top) to which 4-azido-L-phenylalanine is not added. Although not visible, the expression of fluorescent protein was confirmed in the medium (bottom) to which 4-azido-L-phenylalanine was added.
또한, 도 2에서도 확인할 수 있듯이, 재조합 티로실-tRNA 합성효소 JNCC를 갖고 있는 pEVOL-JNCC와 pSEVA631-sfGFP가 클로닝된 재조합 대장균은 4-아지도-L-페닐알라닌이 첨가되지 않은 배지(상)에서는 형광을 보이지 않았지만, 4-아지도-L-페닐알라닌이 첨가된 배지(하)에서는 형광단백질의 발현이 확인되었다.In addition, as can be seen in Figure 2, the recombinant Escherichia coli cloned with pEVOL-JNCC and pSEVA631-sfGFP having recombinant tyrosyl-tRNA synthetase JNCC was not added to 4-azido-L-phenylalanine in the medium (top). Although fluorescence was not observed, expression of fluorescent protein was confirmed in the medium (bottom) to which 4-azido-L-phenylalanine was added.
이러한 결과들로부터 단백질 구조 분석으로부터 설계된 재조합 티로실-tRNA 합성효소 CHI 및 JNCC는 앰버 서프레션 활성과 4-아지도-L-페닐알라닌 결합능을 가지고 있다는 것이 확인되었다.From these results, it was confirmed from protein structure analysis that the designed recombinant tyrosyl-tRNA synthetases CHI and JNCC have amber suppression activity and 4-azido-L-phenylalanine binding ability.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant tyrosyl-tRNA synthetase and method for producing protein using the same <130> PN210427 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 319 <212> PRT <213> Artificial Sequence <220> <223> CHI amino acid sequences <400> 1 Met Asp Lys Leu Glu Leu Val Met Arg Asn Thr Glu Glu Ile Val Thr 1 5 10 15 Val Asp Glu Leu Lys Gly Leu Leu Glu Lys Pro Ser Arg Pro Arg Ala 20 25 30 Thr Ile Gly Phe Glu Pro Ser Gly Lys Val His Leu Gly His Met Leu 35 40 45 Gln Ala Asn Lys Leu Leu Asp Leu Gln Arg Ala Gly Phe Asp Val Val 50 55 60 Val Leu Leu Ala Asp Leu His Ala Phe Leu Asn Glu Lys Gly Thr Leu 65 70 75 80 Glu Glu Val Arg Gln Ile Ala Asp Tyr Asn Arg Asp Cys Phe Met Ala 85 90 95 Leu Gly Leu Asp Pro Glu Arg Thr Glu Phe Val Tyr Gly Thr Asn Phe 100 105 110 Gln Leu Gln Pro Asp Tyr Met Leu Lys Ile Leu Gln Met Ala Arg Asn 115 120 125 Thr Ser Leu Asn Arg Ala Arg Arg Ser Met Asp Glu Ile Ser Arg Asn 130 135 140 Ala Glu Asn Pro Met Val Ser Gln Met Ile Tyr Pro Leu Met Gln Ala 145 150 155 160 Val Pro Leu Ala Asp Gln Lys Ile Asp Leu Ala Val Gly Gly Ile Glu 165 170 175 Gln Arg Lys Ile His Met Leu Ala Arg Glu Glu Leu Pro Arg Leu Gly 180 185 190 Leu Pro Ala Pro Val Cys Leu His Asn Pro Val Ile Pro Gly Leu Asn 195 200 205 Gly Glu Lys Met Ser Ser Ser Lys Gly Asn Phe Ile Ala Val Asp Glu 210 215 220 Pro Ala Gln Asp Val Glu Lys Lys Ile Lys Ser Ala Phe Cys Pro Ala 225 230 235 240 Lys Val Val Glu Asn Asn Pro Val Leu Ala Ile Cys Lys Tyr His Val 245 250 255 Phe Pro Arg Met Glu Val Gly Met Thr Ile Ser Arg Pro Glu Lys Phe 260 265 270 Gly Gly Asp Val His Tyr Ala Ser Tyr Glu Gln Leu Glu Ala Asp Phe 275 280 285 Val Ser Gly Ala Met His Pro Met Asp Leu Lys Lys Ser Cys Ala Glu 290 295 300 Cys Met Ile Glu Ile Leu Ala Pro Val Arg Glu Lys Met Lys Val 305 310 315 <210> 2 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> CHI nucleic sequences <400> 2 agatctatgg acaaactgga attggtcatg cggaacactg aggagatcgt taccgtcgat 60 gaattgaagg gtctgcttga aaaaccgagt cgtccacgcg ccacgattgg ctttgaacct 120 tctggcaaag tacaccttgg gcatatgtta caagcgaaca aacttttaga cttgcagcgc 180 gcggggttcg acgttgtagt tttactggcc gacttgcacg cctttttgaa cgagaaagga 240 actttagaag aggtccggca aattgctgac tataaccggg actgctttat ggcccttggt 300 cttgacccgg agcgcaccga gtttgtgtac ggaacgaact tccaattaca accggattac 360 atgctgaaaa tcttgcagat ggcacggaac acctctctga atagagcacg ccgcagcatg 420 gacgaaataa gcagaaacgc tgagaatcct atggtgagcc agatgatcta tcctttgatg 480 caagcggtac ctcttgccga ccagaagatc gatttagctg tcggaggaat tgaacaacgt 540 aagatccaca tgttagcgcg cgaagaactt cccagattag gtttgcctgc gcctgtgtgt 600 ttgcataacc cagtgattcc tggccttaat ggtgagaaga tgtcatcatc caagggtaac 660 tttatcgcag tggacgaacc cgctcaagat gttgagaaaa agattaaaag tgccttctgt 720 ccggctaaag tagtggaaaa taacccagtg cttgctattt gtaaatatca tgtgttccct 780 cgtatggaag tgggcatgac tattagtcgc cccgaaaagt ttggaggtga tgttcattac 840 gcttcttatg aacaactgga ggcggatttc gtcagcggcg cgatgcaccc aatggacctg 900 aaaaaaagct gtgcggaatg catgatagag attctggccc cggtgagaga aaagatgaaa 960 gtgtaactgc ag 972 <210> 3 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> JNCC amino acid sequences <400> 3 Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr 20 25 30 Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His 145 150 155 160 Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro Val Leu Thr Leu Asn Gly Glu Lys Met Ser Ser Ser Lys Gly 195 200 205 Asn Asn Ile Ala Val Asp Glu Pro Ala Gln Asp Val Glu Lys Lys Ile 210 215 220 Lys Ser Ala Phe Cys Pro Ala Lys Val Val Glu Asn Asn Pro Val Leu 225 230 235 240 Ala Ile Cys Lys Tyr His Val Phe Pro Arg Met Glu Val Gly Met Thr 245 250 255 Ile Ser Arg Pro Glu Lys Phe Gly Gly Asp Val His Tyr Ala Ser Tyr 260 265 270 Glu Gln Leu Glu Ala Asp Phe Val Ser Gly Ala Met His Pro Met Asp 275 280 285 Leu Lys Lys Ser Cys Ala Glu Cys Met Ile Glu Ile Leu Ala Pro Val 290 295 300 Arg Glu Lys Met Lys Val 305 310 <210> 4 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> JNCC nucleic sequences <400> 4 agatctatgg acgagttcga aatgataaag cgtaacacga gcgaaattat ttcagaggaa 60 gaattgagag aagtcttaaa aaaagacgaa aaatctgcga ctataggctt cgagcccagt 120 ggtaagattc atttaggcca ctaccttcag attaaaaaaa tgattgattt gcaaaatgcg 180 ggatttgaca tcattatatt actggccgat ttacatgcct atctgaatca aaaaggagaa 240 ttagatgaga ttcgtaaaat aggcgactac aacaaaaagg tgtttgaagc aatgggcctt 300 aaagcgaaat atgtttacgg gtcgaacttc cagttagata aggattatac acttaatgta 360 tatagacttg cgttaaaaac aaccttaaaa cgtgctagac gttctatgga attaattgca 420 cgcgaggatg agaatccgaa ggtagcagaa gtaatctatc ctatcatgca ggtaaatccc 480 cttcactacc aaggagtcga tgttgcggtc ggtggtatgg aacaacgtaa gatacatatg 540 cttgcccgtg aattattgcc aaagaaggtt gtctgtattc ataacccagt tcttaccctt 600 aacggggaaa agatgtcctc gagcaagggc aataatatcg cggtggatga gcctgcgcaa 660 gatgtagaaa aaaagataaa gtctgcgttc tgtccggcga aggtcgtcga aaacaatcca 720 gtacttgcga tctgcaaata ccacgtattc ccgagaatgg aagtcggaat gaccatcagc 780 agaccagaga agtttggcgg agacgtacac tatgcttcgt acgaacaatt agaggcggac 840 tttgtgtcag gggctatgca tcctatggat ttaaagaagt cttgcgcaga gtgcatgatc 900 gagattcttg cccctgttag agaaaaaatg aaagtgtaac tgcag 945 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CHI forward primer <400> 5 agatctatgg acaaa 15 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CHI reverse primer <400> 6 ctgcagttac acttt 15 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> JNCC forward primer <400> 7 agatctatgg acgag 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> JNCC reverse prime <400> 8 ctgcagttac acttt 15 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant tyrosyl-tRNA synthetase and method for producing protein using the same <130> PN210427 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 319 <212> PRT <213> artificial sequence <220> <223> CHI amino acid sequences <400> 1 Met Asp Lys Leu Glu Leu Val Met Arg Asn Thr Glu Glu Ile Val Thr 1 5 10 15 Val Asp Glu Leu Lys Gly Leu Leu Glu Lys Pro Ser Arg Pro Arg Ala 20 25 30 Thr Ile Gly Phe Glu Pro Ser Gly Lys Val His Leu Gly His Met Leu 35 40 45 Gln Ala Asn Lys Leu Leu Asp Leu Gln Arg Ala Gly Phe Asp Val Val 50 55 60 Val Leu Leu Ala Asp Leu His Ala Phe Leu Asn Glu Lys Gly Thr Leu 65 70 75 80 Glu Glu Val Arg Gln Ile Ala Asp Tyr Asn Arg Asp Cys Phe Met Ala 85 90 95 Leu Gly Leu Asp Pro Glu Arg Thr Glu Phe Val Tyr Gly Thr Asn Phe 100 105 110 Gln Leu Gln Pro Asp Tyr Met Leu Lys Ile Leu Gln Met Ala Arg Asn 115 120 125 Thr Ser Leu Asn Arg Ala Arg Arg Ser Met Asp Glu Ile Ser Arg Asn 130 135 140 Ala Glu Asn Pro Met Val Ser Gln Met Ile Tyr Pro Leu Met Gln Ala 145 150 155 160 Val Pro Leu Ala Asp Gln Lys Ile Asp Leu Ala Val Gly Gly Ile Glu 165 170 175 Gln Arg Lys Ile His Met Leu Ala Arg Glu Glu Leu Pro Arg Leu Gly 180 185 190 Leu Pro Ala Pro Val Cys Leu His Asn Pro Val Ile Pro Gly Leu Asn 195 200 205 Gly Glu Lys Met Ser Ser Ser Lys Gly Asn Phe Ile Ala Val Asp Glu 210 215 220 Pro Ala Gln Asp Val Glu Lys Lys Ile Lys Ser Ala Phe Cys Pro Ala 225 230 235 240 Lys Val Val Glu Asn Asn Pro Val Leu Ala Ile Cys Lys Tyr His Val 245 250 255 Phe Pro Arg Met Glu Val Gly Met Thr Ile Ser Arg Pro Glu Lys Phe 260 265 270 Gly Gly Asp Val His Tyr Ala Ser Tyr Glu Gln Leu Glu Ala Asp Phe 275 280 285 Val Ser Gly Ala Met His Pro Met Asp Leu Lys Lys Ser Cys Ala Glu 290 295 300 Cys Met Ile Glu Ile Leu Ala Pro Val Arg Glu Lys Met Lys Val 305 310 315 <210> 2 <211> 972 <212> DNA <213> artificial sequence <220> <223> CHI nucleic sequences <400> 2 agatctatgg acaaactgga attggtcatg cggaacactg aggagatcgt taccgtcgat 60 gaattgaagg gtctgcttga aaaaccgagt cgtccacgcg ccacgattgg ctttgaacct 120 tctggcaaag tacaccttgg gcatatgtta caagcgaaca aacttttaga cttgcagcgc 180 gcggggttcg acgttgtagt tttactggcc gacttgcacg cctttttgaa cgagaaagga 240 actttagaag aggtccggca aattgctgac tataaccggg actgctttat ggcccttggt 300 cttgacccgg agcgcaccga gtttgtgtac ggaacgaact tccaattaca accggattac 360 atgctgaaaa tcttgcagat ggcacggaac acctctctga atagagcacg ccgcagcatg 420 gacgaaataa gcagaaacgc tgagaatcct atggtgagcc agatgatcta tcctttgatg 480 caagcggtac ctcttgccga ccagaagatc gatttagctg tcggaggaat tgaacaacgt 540 aagatccaca tgttagcgcg cgaagaactt cccagattag gtttgcctgc gcctgtgtgt 600 ttgcataacc cagtgattcc tggccttaat ggtgagaaga tgtcatcatc caagggtaac 660 tttatcgcag tggacgaacc cgctcaagat gttgagaaaa agattaaaag tgccttctgt 720 ccggctaaag tagtggaaaa taacccagtg cttgctattt gtaaatatca tgtgttccct 780 cgtatggaag tgggcatgac tattagtcgc cccgaaaagt ttggaggtga tgttcattac 840 gcttcttatg aacaactgga ggcggatttc gtcagcggcg cgatgcaccc aatggacctg 900 aaaaaaagct gtgcggaatg catgatagag attctggccc cggtgagaga aaagatgaaa 960 gtgtaactgc ag 972 <210> 3 <211> 310 <212> PRT <213> artificial sequence <220> <223> JNCC amino acid sequences <400> 3 Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr 20 25 30 Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His 145 150 155 160 Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro Val Leu Thr Leu Asn Gly Glu Lys Met Ser Ser Ser Lys Gly 195 200 205 Asn Asn Ile Ala Val Asp Glu Pro Ala Gln Asp Val Glu Lys Lys Ile 210 215 220 Lys Ser Ala Phe Cys Pro Ala Lys Val Val Glu Asn Asn Pro Val Leu 225 230 235 240 Ala Ile Cys Lys Tyr His Val Phe Pro Arg Met Glu Val Gly Met Thr 245 250 255 Ile Ser Arg Pro Glu Lys Phe Gly Gly Asp Val His Tyr Ala Ser Tyr 260 265 270 Glu Gln Leu Glu Ala Asp Phe Val Ser Gly Ala Met His Pro Met Asp 275 280 285 Leu Lys Lys Ser Cys Ala Glu Cys Met Ile Glu Ile Leu Ala Pro Val 290 295 300 Arg Glu Lys Met Lys Val 305 310 <210> 4 <211> 945 <212> DNA <213> artificial sequence <220> <223> JNCC nucleic sequences <400> 4 agatctatgg acgagttcga aatgataaag cgtaacacga gcgaaattat ttcagaggaa 60 gaattgagag aagtcttaaa aaaagacgaa aaatctgcga ctataggctt cgagcccagt 120 ggtaagattc atttaggcca ctaccttcag attaaaaaaa tgattgattt gcaaaatgcg 180 ggatttgaca tcattattatt actggccgat ttacatgcct atctgaatca aaaaggagaa 240 ttagatgaga ttcgtaaaat aggcgactac aacaaaaagg tgtttgaagc aatgggcctt 300 aaagcgaaat atgtttacgg gtcgaacttc cagttagata aggattatac acttaatgta 360 tatagacttg cgttaaaaac aaccttaaaa cgtgctagac gttctatgga attaattgca 420 cgcgaggatg agaatccgaa ggtagcagaa gtaatctatc ctatcatgca ggtaaatccc 480 cttcactacc aaggagtcga tgttgcggtc ggtggtatgg aacaacgtaa gatacatatg 540 cttgcccgtg aattattgcc aaagaaggtt gtctgtattc ataacccagt tcttaccctt 600 aacgggggaaa agatgtcctc gagcaagggc aataatatcg cggtggatga gcctgcgcaa 660 gatgtagaaa aaaagataaa gtctgcgttc tgtccggcga aggtcgtcga aaacaatcca 720 gtacttgcga tctgcaaata ccacgtattc ccgagaatgg aagtcggaat gaccatcagc 780 agaccagaga agtttggcgg agacgtacac tatgcttcgt acgaacaatt agaggcggac 840 tttgtgtcag gggctatgca tcctatggat ttaaagaagt cttgcgcaga gtgcatgatc 900 gagattcttg cccctgttag agaaaaaatg aaagtgtaac tgcag 945 <210> 5 <211> 15 <212> DNA <213> artificial sequence <220> <223> CHI forward primer <400> 5 agatctatgg acaaa 15 <210> 6 <211> 15 <212> DNA <213> artificial sequence <220> <223> CHI reverse primer <400> 6 ctgcagttac acttt 15 <210> 7 <211> 15 <212> DNA <213> artificial sequence <220> <223> JNCC forward primer <400> 7 agatctatgg acgag 15 <210> 8 <211> 15 <212> DNA <213> artificial sequence <220> <223> JNCC reverse prime <400> 8 ctgcagttac acttt 15
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