KR20230092461A - Codon optimized atpif1 polynucleotide and recombinant vector comprising the same - Google Patents
Codon optimized atpif1 polynucleotide and recombinant vector comprising the same Download PDFInfo
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Abstract
Description
본 발명은 코돈 최적화된 ATPIF1 폴리뉴클레오티드 및 이를 포함하는 재조합 벡터에 관한 것이다.The present invention relates to a codon-optimized ATPIF1 polynucleotide and a recombinant vector comprising the same.
ATPIF1 (ATP synthase inhibitory factor subunit 1) 유전자로부터 발현되는 IF1 단백질은 미토콘드리아 막에 존재하는 전자전달계의 한 구성요소인 F-type의 ATPase에 결합하여 회전(rotation)을 방해함으로써 ATP의 합성 혹은 분해를 저해하며, 이에 따른 미토콘드리아 기능성 및 세포 생존에 영향을 미치는 것으로 알려져 있다. 이를 토대로 대부분의 연구는 IF1 단백질의 병태생리학적 기능을 규명하기 위해 유전자 조작을 통한 세포 내 IF1의 발현에 중점을 두고 있으나, 인체 내에 분비되어 존재하는 IF1 단백질에 대한 분자생리학적 연구는 진척이 없다.IF1 protein expressed from ATPIF1 (ATP synthase inhibitory factor subunit 1) gene binds to F-type ATPase, a component of the electron transport system present in the mitochondrial membrane, and inhibits rotation by interfering with the synthesis or degradation of ATP. It is known to affect mitochondrial function and cell survival accordingly. Based on this, most studies focus on the expression of IF1 in cells through genetic manipulation to identify the pathophysiological function of IF1 protein, but there is no progress in molecular physiological research on IF1 protein secreted and present in the human body. .
특히 인간 IF1 단백질은 재조합 단백질로 발현시키면 불용성 형태로 발현되는 경우가 많아 연구에 어려움이 있다.In particular, when the human IF1 protein is expressed as a recombinant protein, it is often expressed in an insoluble form, making research difficult.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로 본 발명자들은 IF1 코딩 서열의 약 25.9%를 최적화한 결과 대부분의 재조합 단백질이 수용성 분획에서 발견되는 것을 확인하였다.The present invention was made to solve the above problems in the prior art, and as a result of optimizing about 25.9% of the IF1 coding sequence, the present inventors confirmed that most of the recombinant proteins were found in the water-soluble fraction.
따라서, 본 발명은 IF1 코딩 서열을 코돈 최적화한, 서열번호 1의 서열을 포함하는 폴리뉴클레오티드를 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a polynucleotide comprising the sequence of SEQ ID NO: 1 in which the IF1 coding sequence is codon-optimized.
상기 목적을 달성하기 위하여 본 발명의 일 양상은 ATP 합성효소 억제 인자 서브유닛 1 (ATP synthase inhibitory factor subunit 1, ATPIF1) 코딩 서열을 코돈 최적화한, 서열번호 1의 서열을 포함하는 폴리뉴클레오티드를 제공한다.In order to achieve the above object, one aspect of the present invention provides a polynucleotide comprising the sequence of SEQ ID NO: 1, in which the coding sequence of ATP synthase inhibitory factor subunit 1 (ATPIF1) is codon-optimized. .
본 명세서에서, "ATP 합성효소 억제 인자 서브유닛 1 (ATP synthase inhibitory factor subunit 1, ATPIF1)" 유전자는 ATP 합성효소 억제 인자 서브유닛 1, 즉 IF1 단백질을 코딩하는 유전자이다. IF1 단백질은 미토콘드리아 막에 존재하는 전자전달계의 한 구성요소인 F-type의 ATPase에 결합하여 ATP의 합성 혹은 분해를 저해하며, 이에 따른 미토콘드리아 기능성 및 세포 생존에 영향을 미치는 것으로 알려져 있다.In the present specification, the "ATP synthase inhibitory factor subunit 1 (ATPIF1)" gene is a gene encoding ATP synthase
본 발명자들은 인간 ATPIF1 유전자의 염기서열 (Gene bank accession no: NM_016311.5) 중에서 성숙한 펩타이드(mature peptide) 코딩 서열을 포함하는 136-378 bp (243 bp 길이; 서열번호 4)의 염기서열에 대해 코돈 최적화를 수행하여 약 25.9%의 염기를 교체하였다 (도 1).Among the nucleotide sequences of the human ATPIF1 gene (Gene bank accession no: NM_016311.5), the present inventors found a codon for a nucleotide sequence of 136-378 bp (243 bp length; SEQ ID NO: 4) including a mature peptide coding sequence. Optimization was performed to replace about 25.9% of the bases (FIG. 1).
또한, 본 발명자들은 pH에 의한 영향을 덜 받도록 천연형 IF1 단백질 코딩 서열(서열번호 4)에서 145-147번 염기를 CAT(H)에서 AAG(K)로 변경하여 코돈 최적화한 돌연변이 IF1 서열(서열번호 3) 또한 고안하였다.In addition, the present inventors changed the 145-147 bases from CAT (H) to AAG (K) in the native IF1 protein coding sequence (SEQ ID NO: 4) to be less affected by pH, and the codon-optimized mutant IF1 sequence (sequence No. 3) was also devised.
상기 코돈 최적화된 서열(서열번호 1 내지 3)은 천연형 IF1 단백질 대비 발현량이 우수하고, 수용성 발현 경향 또한 증가하는 장점을 가진다 (도 4 및 5).The codon-optimized sequences (SEQ ID NOs: 1 to 3) have an advantage in that the expression level is superior to that of the wild-type IF1 protein and the water-soluble expression tendency is also increased (Figs. 4 and 5).
본 발명의 일 구체예에 따르면, 상기 서열번호 1의 서열을 포함하는 폴리뉴클레오티드는 서열번호 2 또는 서열번호 3의 서열을 포함한다. 서열번호 1의 서열에서 145-147번 염기는 "MAS"로 M은 시토신(C) 또는 아데닌(A)을 의미하고, S는 시토신(C) 또는 구아닌(G)을 의미한다. 따라서, 서열번호 2의 서열에서 145-147번 염기는 "CAC"이고, 서열번호 3의 서열에서 145-147번 염기는 "AAG"이다.According to one embodiment of the present invention, the polynucleotide comprising the sequence of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In the sequence of SEQ ID NO: 1, bases 145-147 are "MAS", M means cytosine (C) or adenine (A), and S means cytosine (C) or guanine (G). Therefore, bases 145-147 in the sequence of SEQ ID NO: 2 are "CAC", and bases 145-147 in the sequence of SEQ ID NO: 3 are "AAG".
본 발명의 다른 양상은 상기 폴리뉴클레오티드를 포함하는 재조합 벡터를 제공한다.Another aspect of the present invention provides a recombinant vector comprising the polynucleotide.
본 명세서에 있어서, "벡터(vector)"는 세포 내로 전달되는 DNA 단편, 핵산 분자 등을 의미하며, 상기 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재제조될 수 있다. 용어 "전달체"와 호환하여 사용될 수 있다. In the present specification, "vector" means a DNA fragment, a nucleic acid molecule, etc. delivered into a cell, and the vector replicates DNA and can be independently remanufactured in a host cell. May be used interchangeably with the term "delivery vehicle".
용어, "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 본 발명의 재조합 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터, 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 재조합 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 등을 포함할 수 있으며, 목적에 따라 다양하게 제조될 수 있다. 상기 프로모터는 바람직하게는 대장균에서 단백질을 발현하는데 사용되는 프로모터일 수 있으나, 이에 제한되지 않는다.The term "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express the operably linked coding sequence in a particular host organism. Recombinant vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable recombinant vectors may include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, and the like, and may be prepared in various ways depending on the purpose. The promoter may preferably be a promoter used to express a protein in E. coli, but is not limited thereto.
본 명세서에 있어서, "작동가능하게 연결된(operably linked)"이란 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다.As used herein, "operably linked" means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence.
상기 폴리뉴클레오티드와 발현 벡터와의 작동적 연결은 본 발명이 속하는 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.Operational linkage between the polynucleotide and the expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art. can be used.
본 발명의 또 다른 양상은 상기 재조합 벡터가 도입된 재조합 숙주세포를 제공한다.Another aspect of the present invention provides a recombinant host cell into which the recombinant vector is introduced.
본 발명에서, 상기 재조합 숙주세포는 미생물, 곤충세포 및 동물세포로 이루어진 군에서 선택될 수 있으며, 상기 미생물은 세균, 효모 또는 곰팡일 수 있다.In the present invention, the recombinant host cell may be selected from the group consisting of microorganisms, insect cells and animal cells, and the microorganisms may be bacteria, yeast or fungi.
본 발명의 일 구체예에 따르면, 상기 미생물은 대장균일 수 있다.According to one embodiment of the present invention, the microorganism may be Escherichia coli.
숙주세포에 상기 재조합 벡터를 도입하는 방법은 특정의 방법으로 반드시 한정되는 것은 아니고, 통상적으로 당업계에서 널리 사용되는 방법을 사용할 수 있다. 예를 들어, 전기충격 또는 열충격 방법을 사용할 수 있고, 동물세포에 재조합 벡터를 도입할 때는 상업적으로 판매되는 형질주입(transfection) 시약을 사용할 수 있다.A method of introducing the recombinant vector into a host cell is not necessarily limited to a specific method, and a method commonly used in the art may be used. For example, an electric shock or heat shock method may be used, and a commercially available transfection reagent may be used when introducing a recombinant vector into animal cells.
본 발명의 또 다른 양상은 상기 재조합 숙주세포를 배양하는 단계를 포함하는 ATP 합성효소 억제 인자 서브유닛 1의 생산 방법을 제공한다.Another aspect of the present invention provides a method for producing ATP synthase
상기 숙주세포를 배양하는 단계에는 당업계에서 통상적으로 널리 사용되는 방법을 사용할 수 있다. 숙주세포가 곤충세포 및 동물세포이면 상업적으로 판매되는 세포 배양 배지를 사용할 수 있으며, 세포 배양에 필요한 기타 물질을 첨가할 수 있다.본 발명의 일 구체예에 따르면, 상기 생산 방법은 재조합 숙주세포가 재조합 미생물인 경우, 재조합 미생물을 LB 배지(Luria-Bertani broth)에서 배양하는 단계; 및 재조합 미생물이 일정 수준으로 생장하면 재조합 단백질 발현을 유도하는 단계;를 포함할 수 있다. 재조합 단백질의 발현 유도는 통상적으로 당업계에서 널리 사용되는 방법을 사용할 수 있으며, 예를 들어, 배양 배지에 IPTG를 첨가하여 단백질 발현을 유도할 수 있다.In the step of culturing the host cells, methods commonly and widely used in the art may be used. If the host cells are insect cells or animal cells, a commercially available cell culture medium may be used, and other substances necessary for cell culture may be added. In the case of a recombinant microorganism, culturing the recombinant microorganism in LB medium (Luria-Bertani broth); and inducing expression of the recombinant protein when the recombinant microorganism grows to a certain level. Expression of a recombinant protein may be induced using a method widely used in the art, and, for example, protein expression may be induced by adding IPTG to a culture medium.
본 발명의 코돈 최적화된 ATPIF1 폴리뉴클레오티드 및 이를 포함하는 재조합 벡터를 사용하면 ATPIF1 (IF1) 단백질의 발현을 증가시킬 수 있고, 수용성 발현 경향 또한 증가시킬 수 있다.By using the codon-optimized ATPIF1 polynucleotide of the present invention and a recombinant vector containing the same, the expression of ATPIF1 (IF1) protein can be increased, and the water-soluble expression tendency can also be increased.
도 1은 천연형 ATPIF1 유전자 서열, 코돈 최적화된 ATPIF1 유전자 서열 및 코돈 최적화된 ATPIF1 변이형 유전자 서열을 비교, 정렬한 결과를 나타낸다 (Origianal: 천연형 ATPIF1 유전자 서열; 및 Optimized: 코돈 최적화된 ATPIF1 유전자 또는 변이형 유전자 서열).
도 2는 유전자 삽입과 재조합 단백질 생산에 사용한 pET-28a 벡터의 개략적인 구조를 나타낸다.
도 3은 코돈 최적화된 ATPIF1 유전자 또는 변이형 유전자 서열을 포함하는 벡터를 전기영동한 결과이다: 3A는 pET-28a 벡터의 전기영동 결과(M: 전기영동 마커), 3B는 코돈 최적화된 ATPIF1 서열을 포함하는 pET-28a-ATPIF1 최적화 벡터를 그대로(레인 2) 또는 제한효소를 처리한 후(레인 1) 전기영동한 결과, 3C는 코돈 최적화된 변이형 ATPIF1 서열을 포함하는 pET-28a-ATPIF1 변이형 벡터를 그대로(레인 2) 또는 제한효소를 처리한 후(레인 1) 전기영동한 결과, 3D는 pET-28a 벡터(레인 1) 및 천연형 ATPIF1 서열을 포함하는 pET-28a-ATPIF1 벡터의 T7 프로모터 증폭 산물을 전기영동한 결과이다.
도 4는 pET-28a-ATPIF1 최적화 벡터(A), pET-28a-ATPIF1 변이형 벡터(B) 및 pET-28a-ATPIF1 벡터(C)를 대장균에 형질전환시킨 후 단백질 발현 정도를 확인한 결과이다: M-SDS-PAGE 단백질 표준 마커; 1-대장균 파쇄물; 2-대장균 파쇄물의 수용성 분획; 및 3-대장균 파쇄물의 불용성 분획).
도 5는 pET-28a-ATPIF1 최적화 벡터(A), pET-28a-ATPIF1 변이형 벡터(B) 및 pET-28a-ATPIF1 벡터(C)를 대장균에 형질전환 시키고, 상이한 배양 온도 및 IPTG 농도에서 단백질 발현을 유도한 후 단백질 발현 정도를 확인한 결과이다.1 shows the results of comparing and aligning a native ATPIF1 gene sequence, a codon-optimized ATPIF1 gene sequence, and a codon-optimized ATPIF1 variant gene sequence (Origianal: wild-type ATPIF1 gene sequence; and Optimized: codon-optimized ATPIF1 gene or variant gene sequences).
Figure 2 shows a schematic structure of the pET-28a vector used for gene insertion and recombinant protein production.
Figure 3 is the result of electrophoresis of a vector containing the codon-optimized ATPIF1 gene or mutant gene sequence: 3A is the electrophoresis result of the pET-28a vector (M: electrophoretic marker), 3B is the codon-optimized ATPIF1 sequence As a result of electrophoresis of the pET-28a-ATPIF1-optimized vector containing the pET-28a-ATPIF1 vector as it is (lane 2) or after treatment with restriction enzymes (lane 1), 3C is the pET-28a-ATPIF1 variant containing the codon-optimized mutant ATPIF1 sequence. As a result of electrophoresis of the vector as is (lane 2) or after treatment with restriction enzymes (lane 1), 3D was obtained from the pET-28a vector (lane 1) and the T7 promoter of the pET-28a-ATPIF1 vector containing the native ATPIF1 sequence. This is the result of electrophoresis of the amplification product.
Figure 4 shows the result of confirming the protein expression level after transforming the pET-28a-ATPIF1 optimized vector (A), the pET-28a-ATPIF1 mutant vector (B), and the pET-28a-ATPIF1 vector (C) into E. coli: M-SDS-PAGE protein standard marker; 1 - E. coli lysate; 2-aqueous fraction of E. coli lysate; and 3-insoluble fraction of E. coli lysate).
5 is a pET-28a-ATPIF1 optimized vector (A), pET-28a-ATPIF1 mutant vector (B) and pET-28a-ATPIF1 vector (C) transformed into Escherichia coli, protein at different culture temperatures and IPTG concentrations This is the result of confirming the degree of protein expression after inducing expression.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.
실시예 1: 코돈 최적화된 서열을 포함하는 벡터의 제조 및 분석Example 1: Construction and Analysis of Vectors Containing Codon-Optimized Sequences
인간(human) ATPIF1 유전자의 염기서열 (Gene bank accession no: NM_016311.5) 중에서 성숙한 펩타이드(mature peptide) 코딩 서열을 포함하는 136-378 bp (243 bp 길이)의 염기서열에 대해 코돈 최적화를 수행하였다. 변이형 ATPIF1은 이 중 145-147번 서열을 CAT에서 AAG로 변경하여 대장균에서 인간 ATPIF1을 발현하기 위한 코돈 최적화 과정을 수행하였다. 코돈 최적화를 통해 천연형(wild type) ATPIF1 유전자의 염기서열에서 약 25.9%의 염기를 교체하였다 (도 1). Among the base sequences of the human ATPIF1 gene (Gene bank accession no: NM_016311.5), codon optimization was performed on a base sequence of 136-378 bp (243 bp length) including the mature peptide coding sequence. . In the mutant ATPIF1, sequences 145-147 were changed from CAT to AAG, and a codon optimization process was performed to express human ATPIF1 in E. coli. About 25.9% of bases were replaced in the nucleotide sequence of the wild type ATPIF1 gene through codon optimization (FIG. 1).
코돈 최적화 서열을 포함하는 벡터는 pET-28a 벡터의 NdeI과 XhoI 제한효소 부위에 코돈 최적화 서열을 삽입하여 코돈 최적화된 벡터를 제작하였다. 대조군으로 사용한 천연형 인간 ATPIF1 유전자 서열 또한 pET-28a 벡터의 NdeI과 XhoI 제한효소 부위에 삽입하여 벡터를 얻었다.A vector containing the codon-optimized sequence was constructed by inserting the codon-optimized sequence into the NdeI and XhoI restriction enzyme sites of the pET-28a vector. The natural human ATPIF1 gene sequence used as a control was also inserted into the NdeI and XhoI restriction enzyme sites of the pET-28a vector to obtain a vector.
이하에서는 외래 코딩 서열이 도입되지 않은 기본적인 pET-28a 벡터는 "pET-28a 벡터", 천연형 ATPIF1 서열을 포함하는 벡터는 "pET-28a-ATPIF1 벡터", 코돈 최적화된 ATPIF1 서열을 포함하는 벡터는 "pET-28a-ATPIF1 최적화 벡터" 및 코돈 최적화된 변이형 ATPIF1 서열을 포함하는 벡터는 "pET-28a-ATPIF1 변이형 벡터"로 기재한다.Hereinafter, the basic pET-28a vector in which no foreign coding sequence has been introduced is referred to as "pET-28a vector", the vector containing native ATPIF1 sequence is referred to as "pET-28a-ATPIF1 vector", and the vector containing codon-optimized ATPIF1 sequence is referred to as "pET-28a vector". The "pET-28a-ATPIF1 optimized vector" and the vector containing the codon-optimized variant ATPIF1 sequence are described as "pET-28a-ATPIF1 variant vector".
DNA 전기영동을 시행하여 획득한 벡터를 분석하였다. 1% 아가로스 젤(agarose gel)에 DNA 사이즈 마커, pET-28a 벡터, pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터를 로딩한 후 전기영동을 진행하여 벡터 크기를 관찰하였다.The vector obtained by performing DNA electrophoresis was analyzed. After loading the DNA size marker, pET-28a vector, pET-28a-ATPIF1 optimized vector, and pET-28a-ATPIF1 mutant vector on a 1% agarose gel, electrophoresis was performed to observe vector sizes.
또한, pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터에는 MluI과 XhoI 제한효소를 30분 동안 처리한 후 전기영동을 진행하여 정상적인 효소 반응이 일어나는지 추가적으로 확인하였다. pET-28a 벡터와 pET-28a-ATPIF1 벡터는 T7 프로모터 부위를 타겟으로 하는 프라이머로 해당 서열을 증폭하여 추가로 확인하였다. 사용한 프라이머 서열은 다음과 같다: Sense: 5'- TAA TAC GAC TCA CTA TAG G -3' (서열번호 4); Antisense: 5'- GCT AGT TAT TGC TCA GCG G -3' (서열번호 5).In addition, the pET-28a-ATPIF1 optimized vector and the pET-28a-ATPIF1 mutant vector were treated with MluI and XhoI restriction enzymes for 30 minutes, followed by electrophoresis to further confirm whether normal enzymatic reactions occur. The pET-28a vector and the pET-28a-ATPIF1 vector were further confirmed by amplifying the corresponding sequences with primers targeting the T7 promoter region. The primer sequences used were as follows: Sense: 5'- TAA TAC GAC TCA CTA TAG G -3' (SEQ ID NO: 4); Antisense: 5'- GCT AGT TAT TGC TCA GCG G -3' (SEQ ID NO: 5).
전기영동 결과, 5.4 kb 크기인 pET-28a 벡터는 5,000-7,000 bp 사이에서 DNA 밴드가 나타나는 것을 알 수 있었다 (도 3A). 제한효소를 처리하지 않은 pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터 또한 다양한 플라스미드 형태(nick, linear, supercoiled 및 circular form)에 해당하는 밴드를 확인할 수 있었다 (도 3B 및 3C에서 레인 2). 제한효소를 처리한 pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터는 4,000-5,000 bp 사이와 1,000 bp 상단에서 두 개의 밴드를 확인하여 제한효소가 벡터에 작용한 것을 알 수 있었다 (도 3B 및 3C에서 레인 1).As a result of electrophoresis, it was found that the 5.4 kb pET-28a vector showed a DNA band between 5,000 and 7,000 bp (FIG. 3A). The pET-28a-ATPIF1 optimized vector and the pET-28a-ATPIF1 mutant vector, which were not treated with restriction enzymes, were also able to confirm bands corresponding to various plasmid forms (nick, linear, supercoiled and circular forms) (Fig. 3B and 3C lane 2). In the pET-28a-ATPIF1 optimized vector and the pET-28a-ATPIF1 mutant vector treated with restriction enzyme, two bands were identified between 4,000-5,000 bp and at the top of 1,000 bp, indicating that the restriction enzyme acted on the vector (
또한, T7 프로모터 부위의 증폭 산물을 확인한 결과 pET-28a 벡터에서는 T7 프로모터에 해당하는 밴드를 400-500 bp 사이에서 확인할 수 있었고 (도 3D에서 레인 1), pET-28a-ATPIF1 벡터에서는 T7 프로모터+ATPIF1 코딩 서열에 해당하는 밴드를 확인할 수 있었다 (도 3D에서 레인 2)In addition, as a result of confirming the amplification product of the T7 promoter region, a band corresponding to the T7 promoter was confirmed between 400 and 500 bp in the pET-28a vector (
실시예 2: 형질전환 대장균 세포의 제조 및 재조합 단백질의 제조Example 2: Production of transformed E. coli cells and production of recombinant proteins
실시예 1에서 제작한 pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터와 pET-28a-ATPIF1 벡터(대조군)를 재조합 단백질 발현용 대장균 균주인 BL-21(DE3)과 BL-21-Codon Plus(DE3)-RIPL에 삽입하여 형질전환을 시행하였다.The pET-28a-ATPIF1 optimized vector, pET-28a-ATPIF1 mutant vector, and pET-28a-ATPIF1 vector (control) prepared in Example 1 were used for recombinant protein expression in E. coli strains BL-21(DE3) and BL-21 Transformation was performed by inserting into -Codon Plus (DE3)-RIPL.
각 대장균 균주를 LB 배지에서 37℃로 2시간 동안 배양하고, 1 mM의 IPTG (isopropyl-β-D-thiogalactopyranoside)를 처리하여 25℃에서 12시간 추가로 배양하였다. 재조합 단백질을 포함한 세포는 먼저 세포 파쇄 (sonication) 과정과 원심분리를 통해 수용성 분획과 불용성 분획으로 분리하였고, 각 분획을 4-20% SDS (sodium dodecyl sulfate) 젤에 로딩하여 단백질 발현 정도를 확인하였다. 단백질 분자량은 킬로달톤(kilodaltons; kDa)으로 표시하였다.Each E. coli strain was cultured in LB medium at 37° C. for 2 hours, treated with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and further cultured at 25° C. for 12 hours. Cells containing recombinant proteins were first separated into soluble and insoluble fractions through cell disruption (sonication) and centrifugation, and each fraction was loaded on a 4-20% SDS (sodium dodecyl sulfate) gel to confirm the level of protein expression. . Protein molecular weight was expressed in kilodaltons (kDa).
확인 결과, pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터에서 발현된 단백질은 대부분 수용성 분획에서 관찰되었으나 (도 4A 및 4B), pET-28a-ATPIF1 벡터에서 발현된 단백질은 대부분 불용성 분획에서 관찰되었다 (도 4C). 이 결과는 코돈 최적화에 의해 재조합 단백질의 수용성 발현 경향이 증가되었다는 것을 의미한다.As a result, most of the proteins expressed in the pET-28a-ATPIF1 optimized vector and the pET-28a-ATPIF1 mutant vector were observed in the soluble fraction (FIGS. 4A and 4B), but most of the proteins expressed in the pET-28a-ATPIF1 vector were insoluble. fraction was observed (Fig. 4C). This result means that the water-soluble expression tendency of the recombinant protein was increased by codon optimization.
도 4에서 각 레인은 다음을 의미한다: M-SDS-PAGE 단백질 표준 마커; 1-대장균 파쇄물; 2-대장균 파쇄물의 수용성 분획; 및 3-대장균 파쇄물의 불용성 분획.In Figure 4, each lane means: M-SDS-PAGE protein standard marker; 1 - E. coli lysate; 2-aqueous fraction of E. coli lysate; and 3-insoluble fraction of E. coli lysate.
또한, 코돈 최적화 유무에 따른 단백질의 발현량 변화를 상이한 배양 온도 및 IPTG 농도에서 확인하였다. 그 결과, IPTG를 첨가한 모든 샘플에서 농도와 무관하게 pET-28a-ATPIF1 벡터(도 4C)와 비교하여 pET-28a-ATPIF1 최적화 벡터 및 pET-28a-ATPIF1 변이형 벡터에서 발현된 단백질의 발현량(각각 도 4A 및 4B)이 높은 것을 알 수 있었다. 구체적으로, 코돈 최적화에 따라 단백질 발현량이 약 89% 증가한 것을 관찰하여 코돈 최적화에 의한 재조합 단백질의 발현도 증가를 확인하였다.In addition, changes in protein expression levels with and without codon optimization were confirmed at different culture temperatures and IPTG concentrations. As a result, the expression level of proteins expressed in the pET-28a-ATPIF1 optimized vector and the pET-28a-ATPIF1 mutant vector compared to the pET-28a-ATPIF1 vector (FIG. 4C) regardless of the concentration in all samples to which IPTG was added. (Fig. 4A and 4B, respectively) was found to be high. Specifically, it was observed that the protein expression level increased by about 89% according to the codon optimization, confirming the increase in the expression level of the recombinant protein due to the codon optimization.
<110> Korea University Research and Business Foundation MEDI&GENE <120> CODON OPTIMIZED ATPIF1 POLYNUCLEOTIDE AND RECOMBINANT VECTOR COMPRISING THE SAME <130> P21U13C1653 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> codon optimized ATPIF1 <400> 1 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acacmasgaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 2 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> codon optimized ATPIF1 <400> 2 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acaccacgaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 3 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> codon optimized mutated ATPIF1 <400> 3 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acacaaggaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 4 <211> 245 <212> DNA <213> Homo sapiens <400> 4 ggctcggatc agtccgagaa tgtcgaccgg ggcgcgggct ccatccggga agccggtggg 60 gccttcggaa agagagagca ggctgaagag gaacgatatt tccgagcaca gagtagagaa 120 caactggcag ctttgaaaaa acaccatgaa gaaagaaatc gttcatcata agaaggagat 180 tgagcgtctg cagaaagaaa ttgagcgcca taaagcagaa gatcaaaatg ctaaaacatg 240 atgat 245 <110> Korea University Research and Business Foundation MEDI&GENE <120> CODON OPTIMIZED ATPIF1 POLYNUCLEOTIDE AND RECOMBINANT VECTOR COMPRISING THE SAME <130> P21U13C1653 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 243 <212> DNA <213> artificial sequence <220> <223> codon optimized ATPIF1 <400> 1 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acacmasgaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 2 <211> 243 <212> DNA <213> artificial sequence <220> <223> codon optimized ATPIF1 <400> 2 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acaccacgaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 3 <211> 243 <212> DNA <213> artificial sequence <220> <223> codon optimized mutated ATPIF1 <400> 3 ggtagcgacc agagcgagaa cgtggatcgt cgtgcgggca gcattcgtga ggcgggtggc 60 gcgtttggta aacgtgaaca agcggaggaa gagcgttact ttcgtgcgca gagccgtgaa 120 caactggcgg cgctgaagaa acacaaggaa gaggaaatcg ttcaccacaa gaaagagatt 180 gaacgtctgc agaaagagat cgaacgtcac aagcaaaaaa ttaagatgct gaagcacgac 240 gat 243 <210> 4 <211> 245 <212> DNA <213> Homo sapiens <400> 4 ggctcggatc agtccgagaa tgtcgaccgg ggcgcgggct ccatccggga agccggtggg 60 gccttcggaa agagagagca ggctgaagag gaacgatatt tccgagcaca gagtagagaa 120 caactggcag ctttgaaaaa acaccatgaa gaaagaaatc gttcatcata agaaggagat 180 tgagcgtctg cagaaagaaa ttgagcgcca taaagcagaa gatcaaaatg ctaaaacatg 240 atgat 245
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