KR20240060975A - A method of manufacturing recombinant sod3 protein - Google Patents
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- 239000010949 copper Substances 0.000 description 4
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Abstract
본 발명은 재조합 SOD3 단백질의 생산 방법에 관한 것이다. HBD partial deletion (CKAA deletion)은 SOD3-WT과 비교시, homogeneous tetramer 형태의 SOD3 생산이 가능하고, 또한 발현율은 약2배 향상시킬 수 있으며, enzyme activity를 유지시킨다. 또한, R213A, R213G, R213L mutants를 도입함으로써, 기존의 proteolytic cleavage (Furin)로 인한 HBD deletion도 억제할 수 있다. HSA fusion HBD partial deletion(CKAA deletion)은 HSA fusion SOD3-WT과 비교시, Homogeneous tetramer 형태의 SOD3 생산이 가능하고, 또한 발현율은 약 2배 향상시킬 수 있으며, Enzyme activity도 잘 유지시킬 수 있다. The present invention relates to a method for producing recombinant SOD3 protein. Compared to SOD3-WT, HBD partial deletion (CKAA deletion) can produce SOD3 in a homogeneous tetramer form, can increase the expression rate by about 2-fold, and maintain enzyme activity. In addition, by introducing R213A, R213G, and R213L mutants, HBD deletion caused by existing proteolytic cleavage (Furin) can be suppressed. Compared to HSA fusion SOD3-WT, HSA fusion HBD partial deletion (CKAA deletion) can produce SOD3 in the form of a homogeneous tetramer, can increase the expression rate by about 2 times, and can maintain enzyme activity well.
Description
본 발명은 재조합 SOD3 단백질의 생산 방법에 관한 것이다.The present invention relates to a method for producing recombinant SOD3 protein.
사람의 슈퍼옥사이드 디스뮤타제(superoxide dismutase ; SOD)는 3가지 유형이 있다. 사람의 SOD1은 세포의 세포질에 이량체(dimer) 형태로 구리 및 아연이 함유되어 기능을 발현하며 구리/아연 에스오디(Cu/Zn SOD)라 불리우고 SOD1 유전자 다형성 중에서 그 어떤 돌연변이체는 근위축성측삭경화증 (amyotrophic lateral sclerosis ; ALS)과 관련되어 있다. 그리고 세포의 미토콘드리아에 존재하는 SOD2는 망간 에스오디(Mn SOD)라 불리면서 사량체(tetramer) 형태로 망간이 함유되는 특성을 가지고 있다. SOD3은 세포 바깥족(extra cellular)에서 사량체(tetramer) 형태로 구리와 아연이 함유되어 사람의 여러신체 부위에서 항산화 역할을 한다.There are three types of human superoxide dismutase (SOD). Human SOD1 expresses its function by containing copper and zinc in the form of a dimer in the cytoplasm of the cell, and is called copper/zinc SOD (Cu/Zn SOD). Among the SOD1 gene polymorphisms, any mutant causes amyotrophic lateral lesions. It is associated with amyotrophic lateral sclerosis (ALS). And SOD2, which exists in the mitochondria of cells, is called manganese SOD (Mn SOD) and has the characteristic of containing manganese in the form of a tetramer. SOD3 contains copper and zinc in the form of a tetramer in the extra cellular region and plays an antioxidant role in various parts of the human body.
SOD3(Extracellular superoxide dismutase 3) 단백질은 tetramer로 구성되어 활성을 띄는 것으로 보고가 되어있다. 하지만 해당 단백질은 non-specific interaction 및 disulfide bond로 인하여 High oligomerization이 유도되고, 또한 SOD3의 C-말단의 proteolytic cleavage로 인하여 heterogeneous SOD3가 유도되는 문제점을 가지고 있다. It has been reported that SOD3 (Extracellular superoxide dismutase 3) protein is composed of a tetramer and is active. However, this protein has the problem of inducing high oligomerization due to non-specific interactions and disulfide bonds, and also inducing heterogeneous SOD3 due to proteolytic cleavage of the C-terminus of SOD3.
SOD3의 독립 발현 시, 낮은 발현율로 인해 의약품 개발에 한계를 갖는다. 이에, K41N mutant는 glycosylation을 유도하여 SOD3의 발현율 향상을 유도하는 등 발현율을 향상하기 위한 다양한 연구가 보고되어 있다. 또한 SOD3는 half-life가 짧아 의약품으로써의 개발에 한계가 있고, 이에 HSA를 fusion하여 해결하는 방법으로 연구가 진행되고 있다.When SOD3 is expressed independently, drug development is limited due to the low expression rate. Accordingly, various studies have been reported to improve the expression rate of SOD3 by inducing glycosylation in the K41N mutant. In addition, SOD3 has a short half-life, which limits its development as a medicine, and research is underway to solve this problem by fusion with HSA.
선행기술문헌을 살펴보면 다음과 같다. Looking at the prior art literature, it is as follows.
특허문헌 1 "EC SOD 및 세포 침투성 EC SOD와 이들의 용도"에는 EC SOD 단백질을 유효성분으로 포함하는 피부질환의 예방 또는 치료용 약학적 조성물이 기재되어 있다, (청구항 1) 또한, 인간 EC SOD에서 헤파린 도메인이 제거된 △HD/EC SOD 단백질과 HIV-1 Tat를 융합시킨 융합 단백질을 제조하여 실험에 이용하는 것이 기재되어 있다.(식별번호 [129]) 특허문헌 2 "세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD) 단백질의 세포 투과성을 증가시키는 방법"에는 헤파린 결합 도메인을 포함하는 EC-SOD 단백질과 아연 2가 양이온을 세포에 접촉시키는 단계를 포함하는 EC-SOD 단백질의 세포 투과성을 증가시키는 방법이 기재되어 있다.(청구항 1) 특허문헌 3에는 "SOD3를 함유하는 건성안의 예방 또는 치료용 조성물"이 기재되어 있다. 살펴보면, C-말단 영역(아미노산 잔기 210번~215번)에 헤파린 결합 도메인(heparin binding domain)을 가지고 있는 SOD3 단백질을 대상으로 하고 있음을 알 수 있다.(식별번호 [0023])Patent Document 1 “EC SOD and Cell-Permeable EC SOD and Their Uses” describes a pharmaceutical composition for preventing or treating skin diseases containing EC SOD protein as an active ingredient (Claim 1). Additionally, human EC SOD It is described that a fusion protein fusing the △HD/EC SOD protein with the heparin domain removed and HIV-1 Tat was prepared and used in experiments. (Identification number [129]) Patent Document 2 “Extracellular secretion superoxide dispersion” “Method for increasing cell permeability of a mutase (EC-SOD) protein” includes contacting a cell with an EC-SOD protein containing a heparin binding domain and a zinc divalent cation. A method for doing so is described. (Claim 1) Patent Document 3 describes “a composition for preventing or treating dry eyes containing SOD3.” Upon examination, it can be seen that the target is the SOD3 protein, which has a heparin binding domain in the C-terminal region (amino acid residues 210 to 215) (identification number [0023]).
특허문헌 4의 청구항 1에는 "HSA(human serum albumin)를 유효성분으로 포함하는 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD) 단백질 촉매활성 안정화용 조성물로서, 상기 조성물은 EC-SOD와 혼합되는 것을 특징으로 하는 조성물"이 기재되어 있다. 청구항 4에서는 청구항 1의 EC-SOD 가 인간 EC-SOD 유전자인 것을 기재하고 있다. 따라서 본 문헌에서는 HSA와 퓨젼되는 대상으로서의 EC-SOD가 인간 유전자이기 때문에 EC SOD의 헤파린 결합 도메인 변경이나 제거를 의도했다고 볼 수 없다.Claim 1 of Patent Document 4 states, “A composition for stabilizing the catalytic activity of extracellularly secreted superoxide dismutase (EC-SOD) protein containing HSA (human serum albumin) as an active ingredient, wherein the composition is mixed with EC-SOD. A “composition characterized by the following” is described. Claim 4 describes that the EC-SOD of claim 1 is a human EC-SOD gene. Therefore, in this document, since EC-SOD, which is the subject of fusion with HSA, is a human gene, it cannot be said that it was intended to change or remove the heparin binding domain of EC SOD.
특허문헌 5는 식물에서 발현이 최적화되도록 조정된 EC-SOD 유전자에 대한 것이다. 살펴보면, 서열번호 2는 표시되는 담배가 선호하는 코돈으로 변환된 SP와 HBD가 제거된 hEC-SOD의 염기서열이다.(식별번호 [0093]) 특허문헌 6의 청구항 1에는 SOD3를 과발현하는 중간엽 줄기세포가 기재되어 있다. 특허문헌 7의 청구항 1에는 슈퍼옥사이드 디스무타아제 3(superoxide dismutase 3, SOD3)를 과발현시킨 줄기세포 유래 세포외 소낭이 기재되어 있다. 특허문헌 8의 청구항 1에는 "BSA(bovine serum albumin) 및 FBS(fetal bovine serum)로 이루어진 군에서 선택되는 하나 이상을 유효성분으로 포함하는 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD) 단백질 안정화용 조성물로서, 상기 조성물은 EC-SOD와 혼합되는 것을 특징으로 하는 조성물."이 기재되어 있다. Patent Document 5 is about the EC-SOD gene adjusted to optimize expression in plants. Looking at it, SEQ ID NO: 2 is the base sequence of hEC-SOD from which SP and HBD converted to the codon preferred by tobacco are removed (identification number [0093]). In claim 1 of Patent Document 6, there is a mesenchyme overexpressing SOD3. Stem cells are described. Claim 1 of Patent Document 7 describes extracellular vesicles derived from stem cells overexpressing superoxide dismutase 3 (SOD3). Claim 1 of Patent Document 8 states, “Extracellularly secreted superoxide dismutase (EC-SOD) protein stabilization containing at least one selected from the group consisting of BSA (bovine serum albumin) and FBS (fetal bovine serum) as an active ingredient. A composition for use, wherein the composition is mixed with EC-SOD."
특허문헌 9에는 헤파린 결합 도메인이 제거된 재조합 EC SOD(extracellular superoxide dismutase) 단백질을 유효성분으로 하는 혈관신생에 의한 질병의 예방 또는 치료용 조성물이 기재되어 있다.Patent Document 9 describes a composition for preventing or treating diseases caused by angiogenesis, which uses a recombinant EC SOD (extracellular superoxide dismutase) protein from which the heparin-binding domain has been removed as an active ingredient.
특허문헌 10의 청구항 1에는 "세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD) 단백질의 N-말단 또는 C-말단에 HSA(human serum albumin) 단백질을 접합시키는 단계를 포함하는 세포외 분비 슈퍼옥사이드 디스뮤테이즈(EC-SOD) 단백질의 안정화 방법"이 기재되어 있다. EC-SOD에 대한 기재를 살펴보면, 기본적으로 C-말단 영역(아미노산 잔기 210번~215번)에 헤파린 결합 도메인(heparin binding domain)을 가지고 있는 것으로 기재하고 있으며(식별번호 [0047]), 또한 청구항 3에 기재된 서열번호 9, 서열번호 22는 각각 서열번호 5의 19-240 fragment, 서열번호 7의 19-240 fragment이기 때문에 헤파린 결합 도메인을 포함하게 된다.(식별번호 [0048], 서열목록) Claim 1 of Patent Document 10 states, “Extracellularly secreted superoxide comprising the step of conjugating HSA (human serum albumin) protein to the N-terminus or C-terminus of the extracellularly secreted superoxide dismutase (EC-SOD) protein. “Method for stabilizing dismutase (EC-SOD) protein” is described. Looking at the description of EC-SOD, it is basically described as having a heparin binding domain in the C-terminal region (amino acid residues 210 to 215) (identification number [0047]), and also in the claims. SEQ ID NO: 9 and SEQ ID NO: 22 described in 3 contain a heparin binding domain because they are the 19-240 fragment of SEQ ID NO: 5 and the 19-240 fragment of SEQ ID NO: 7, respectively. (ID: [0048], Sequence Listing)
해결과제는, SOD3 단백질 생산시 발생되는 High oligomerization, proteolytic cleavage, 낮은 발현율을 해결하는 것이다.The challenge is to solve the high oligomerization, proteolytic cleavage, and low expression rate that occur during SOD3 protein production.
해결수단 1은, 세포외 분비 슈퍼옥사이드 디스뮤테이즈(SOD3, Extracellular superoxide dismutase 3) 단백질의 헤파린 결합 도메인(HBD, Heparin binding domain) 중 219-229 아미노잔 잔기를 제거하는 단계를 포함하는, 재조합 SOD3 단백질의 생산 방법이다.Solution 1 is recombinant SOD3, which includes the step of removing amino acid residues 219-229 of the heparin binding domain (HBD) of the extracellular superoxide dismutase 3 (SOD3) protein. It is a method of producing protein.
해결수단 2는, 상기 해결수단 1에서, R213G, R213A 또는 R213L 변이 중 어느 하나의 변이를 일으키는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법이다.Solution 2 is a method for producing a recombinant SOD3 protein in Solution 1, which further includes the step of causing any one of the R213G, R213A, or R213L mutations.
해결수단 3은, 상기 해결수단 1에서, HSA(Human serum albumin)를 접합하는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법이다.Solution 3 is a method for producing recombinant SOD3 protein in Solution 1, which further includes the step of conjugating HSA (Human serum albumin).
해결수단 4는, 상기 해결수단 2에서, HSA(Human serum albumin)를 접합하는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법이다.Solution 4 is a method for producing recombinant SOD3 protein in Solution 2, which further includes the step of conjugating HSA (Human serum albumin).
해결수단 5는, 상기 해결수단 3에서, R213G, R213A 또는 R213L 변이 중 어느 하나의 변이를 일으키는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법이다.Solution 5 is a method for producing a recombinant SOD3 protein in Solution 3, which further includes the step of causing any one of the R213G, R213A, or R213L mutations.
해결수단 6은, 상기 해결수단 1 내지 5 중 어느 하나의 방법에 의하여 생산된 재조합 SOD3 단백질이다.Solution 6 is a recombinant SOD3 protein produced by any one of solutions 1 to 5 above.
해결수단 7은, 세포외 분비 슈퍼옥사이드 디스뮤테이즈(SOD3, Extracellular superoxide dismutase 3) 단백질의 헤파린 결합 도메인(HBD, Heparin binding domain) 중 219-229 아미노잔 잔기를 제거하는 단계를 포함하는, 재조합 SOD3 단백질의 발현 촉진 방법이다.Solution 7 is recombinant SOD3, which includes the step of removing amino residues 219-229 of the heparin binding domain (HBD) of the extracellular superoxide dismutase 3 (SOD3) protein. It is a method of promoting protein expression.
해결수단 8은, 세포외 분비 슈퍼옥사이드 디스뮤테이즈(SOD3, Extracellular superoxide dismutase 3) 단백질의 헤파린 결합 도메인(HBD, Heparin binding domain) 중 219-229 아미노잔 잔기를 제거하는 단계를 포함하는, 재조합 SOD3 단백질의 안정화 방법이다.Solution 8 is recombinant SOD3, which includes the step of removing amino acid residues 219-229 of the heparin binding domain (HBD) of the extracellular superoxide dismutase 3 (SOD3) protein. It is a method of stabilizing proteins.
본 발명의 효과는 다음과 같다 : HBD partial deletion (CKAA deletion)은 SOD3-WT과 비교시, homogeneous tetramer 형태의 SOD3 생산이 가능하고, 또한 발현율은 약2배 향상시킬 수 있으며, enzyme activity를 유지시킨다. 또한, R213A, R213G, R213L mutants를 도입함으로써, 기존의 proteolytic cleavage (Furin)로 인한 HBD deletion도 억제할 수 있다.The effects of the present invention are as follows: HBD partial deletion (CKAA deletion) is capable of producing SOD3 in a homogeneous tetramer form compared to SOD3-WT, and can also improve the expression rate by about two times and maintain enzyme activity. . In addition, by introducing R213A, R213G, and R213L mutants, HBD deletion caused by existing proteolytic cleavage (Furin) can be suppressed.
본 발명의 효과는 다음과 같다 : HSA fusion HBD partial deletion(CKAA deletion)은 HSA fusion SOD3-WT과 비교시, Homogeneous tetramer 형태의 SOD3 생산이 가능하고, 또한 발현율은 약 2배 향상시킬 수 있으며, Enzyme activity도 잘 유지시킬 수 있다. The effects of the present invention are as follows: HSA fusion HBD partial deletion (CKAA deletion) is capable of producing SOD3 in the form of a homogeneous tetramer when compared to HSA fusion SOD3-WT, and the expression rate can be improved by about 2 times, and the enzyme Activity can also be maintained well.
도 1 및 2는 SOD3-CKAA deletion의 Expression test 결과를 나타내는 그래프이다. 도 3은 SOD3-CKAA deletion의 Tetramer formation test 결과를 나타내는 그래프이다. 도 4는 SOD3-CKAA deletion의 Proteolytic cleavage inhibition test 결과를 나타내는 그래프이다. 도 5는 SOD3-CKAA deletion의 Enzyme activity test 결과를 나타내는 그래프이다.
도 6 및 7은 HSA-SOD3-CKAA deletion의 Expression test 결과를 나타내는 그래프이다. 도 8은 HSA-SOD3-CKAA deletion의 Tetramer formation test 결과를 나타내는 그래프이다. 도 9 및 10은 HSA-SOD3-CKAA deletion의 Enzyme activity test 결과를 나타내는 그래프이다.Figures 1 and 2 are graphs showing the results of the expression test of SOD3-CKAA deletion. Figure 3 is a graph showing the results of the tetramer formation test of SOD3-CKAA deletion. Figure 4 is a graph showing the results of the Proteolytic cleavage inhibition test of SOD3-CKAA deletion. Figure 5 is a graph showing the enzyme activity test results of SOD3-CKAA deletion.
Figures 6 and 7 are graphs showing the results of the expression test of HSA-SOD3-CKAA deletion. Figure 8 is a graph showing the results of the tetramer formation test of HSA-SOD3-CKAA deletion. Figures 9 and 10 are graphs showing the enzyme activity test results of HSA-SOD3-CKAA deletion.
본 명세서 및 청구 범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 안되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.Terms or words used in this specification and claims should not be construed as limited to their ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his or her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on principles.
따라서 본 명세서에 기재된 실험, 실시예 및 도면에 기술된 사항은 본 발명의 가장 바람직한 일 예에 불과할뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다.Accordingly, the matters described in the experiments, examples, and drawings described in this specification are only the most preferred examples of the present invention and do not represent the entire technical idea of the present invention, so at the time of filing the present application, various alternatives may be used to replace them. It should be understood that equivalents and variations may exist.
실시예 1. SOD3-CKAA deletion Construction and TestExample 1. SOD3-CKAA deletion Construction and Test
1) SOD3-CKAA deletion Construction1) SOD3-CKAA deletion construction
하기의 표 1을 보면, SOD3 서열정보가 표시되어 있다. SOD3의 C-말단 HBD(Heparin binding domain) "RKKRRRESECKAA" , 그 뒤의 Linker "GGGGS", 그 뒤의 Histag "HHHHHH"를 확인할 수 있다.Looking at Table 1 below, SOD3 sequence information is displayed. You can check the C-terminal HBD (Heparin binding domain) of SOD3, "RKKRRRESECKAA", followed by Linker "GGGGS", and followed by Histag "HHHHHH".
① partial HBD : CKAA deletion① partial HBD: CKAA deletion
; HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"가 갖는 partial HBD SOD3가 형성된다.; A partial HBD SOD3, which has "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of the HBD (Heparin binding domain) removed, is formed.
② partial HBD - point mutation : CKAA deletion - R213G② partial HBD - point mutation: CKAA deletion - R213G
; HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK G RRESE"가 형성된 partial HBD & point mutation SOD3가 형성된다.; "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed, is treated as R213 mutant to form partial HBD & point mutant SOD3, which is "RKK G RRESE" .
③ partial HBD - point mutation : CKAA deletion - R213A③ partial HBD - point mutation: CKAA deletion - R213A
; HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK A RRESE"가 형성된 partial HBD & point mutation SOD3가 형성된다.; A partial HBD & point mutant SOD3 is formed by processing R213 mutant in "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed, to form "RKK A RRESE" .
④ partial HBD - point mutation : CKAA deletion - R213L ④ partial HBD - point mutation: CKAA deletion - R213L
; HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK L RRESE"가 형성된 partial HBD & point mutation SOD3가 형성된다.; "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed, is treated as R213 mutant to form partial HBD & point mutant SOD3, which is "RKK L RRESE" .
-R213GSOD3-CKAA deletion
-R213G
-R213ASOD3-CKAA deletion
-R213A
-R213LSOD3-CKAA deletion
-R213L
2) SOD3-CKAA deletion Test2) SOD3-CKAA deletion test
① Expression test ① Expression test
도 1 및 2는 SOD3-CKAA deletion의 Expression test 결과를 나타내는 그래프이다. 도 1은 SDS-PAGE 분석 그래프이고, 도 2는 단백질 발현 수율 분석 그래프이다. Figures 1 and 2 are graphs showing the results of the expression test of SOD3-CKAA deletion. Figure 1 is a SDS-PAGE analysis graph, and Figure 2 is a protein expression yield analysis graph.
살펴보면, SOD3-WT 에서는 high oligomerization 패턴이 확인되었다. 그러나 SOD3-HBD_deletion(HBD 완전 제거) 및 partial deletion에서는 High oligomerization이 확인되지 않았다. 또한 Partial deletion construction은 약 2배 이상 발현율이 향상되는 것을 확인하였다. Upon examination, a high oligomerization pattern was confirmed in SOD3-WT. However, high oligomerization was not confirmed in SOD3-HBD_deletion (complete HBD removal) and partial deletion. In addition, it was confirmed that partial deletion construction increases the expression rate by more than two times .
② Tetramer formation test② Tetramer formation test
도 3은 SOD3-CKAA deletion의 Tetramer formation test 결과를 나타내는 그래프이다. 도 3은 SE-HPLC 분석 그래프이다. Figure 3 is a graph showing the results of the tetramer formation test of SOD3-CKAA deletion. Figure 3 is a SE-HPLC analysis graph.
살펴보면, SOD3-WT에서는 high oligomerization 패턴이 확인되었으나, SOD3-HBD_deletion 및 partial deletion에서는 High oligomerization 확인되지 않고, homogenous tetramer가 형성되는 것을 확인하였다. Upon examination, a high oligomerization pattern was confirmed in SOD3-WT, but high oligomerization was not confirmed in SOD3-HBD_deletion and partial deletion, and it was confirmed that a homogenous tetramer was formed .
③ Proteolytic cleavage inhibition test③ Proteolytic cleavage inhibition test
도 4는 SOD3-CKAA deletion의 Proteolytic cleavage inhibition test 결과를 나타내는 그래프이다. Figure 4 is a graph showing the results of the Proteolytic cleavage inhibition test of SOD3-CKAA deletion.
SOD3의 HBD는 Furin 에 의해 proteolytic cleavage 가 일어난다. 이에 따라 재조합 SOD3 생산 시, heterogeneous SOD3가 생산된다. R213G는 Furin cleavage를 저해하는 mutant로 보고가 되었다. (비특허 1 참조)The HBD of SOD3 undergoes proteolytic cleavage by Furin. Accordingly, when recombinant SOD3 is produced, heterogeneous SOD3 is produced. R213G was reported as a mutant that inhibits furin cleavage. (See Non-Patent 1)
살펴보면, In silico library를 통해 도입한 R213L, R213A도 Furin cleavage가 일어나지 않음을 확인하였다. Upon examination, it was confirmed that furin cleavage did not occur in R213L and R213A introduced through the in silico library .
④ Enzyme activity test④ Enzyme activity test
도 5는 SOD3-CKAA deletion의 Enzyme activity test 결과를 나타내는 그래프이다. Figure 5 is a graph showing the enzyme activity test results of SOD3-CKAA deletion.
살펴보면, SOD3-HBD deletion construct 는 활성이 약 70% 감소되었으나, SOD3-CKAA deletion variants 는 SOD3-WT과 유사 활성 갖는 것을 확인하였다. Upon examination, the activity of the SOD3-HBD deletion construct was reduced by about 70%, but the SOD3-CKAA deletion variants were confirmed to have similar activity to SOD3-WT .
3) 평가 분석 고찰3) Evaluation analysis consideration
SOD3는 tetramer를 형성함으로써, 기능을 유지하는 것으로 알려져 있다. SOD3의 HBD (Heparin binding domain)는 heparin binding과 disulfide bridge를 통한 SOD3 interaction의 기능을 갖는다.SOD3 is known to maintain its function by forming a tetramer. The HBD (Heparin binding domain) of SOD3 has the function of heparin binding and SOD3 interaction through a disulfide bridge.
그러나 재조합 SOD3를 생산함에 있어, HBD는 disulfide bridge로 인한 high oligomerization을 유도 하여 heterogeneous protein이 생산된다. 또한, proteolytic cleavage (Furin)로 인한 HBD deletion으로 heterogeneous protein이 생산된다. 해당 문제를 해결하기 위해, HBD deletion construct와 HBD partial deletion (CKAA deletion) constructs를 디자인하였다.However, when producing recombinant SOD3, HBD induces high oligomerization due to disulfide bridge, resulting in the production of heterogeneous protein. Additionally, heterogeneous proteins are produced due to HBD deletion due to proteolytic cleavage (Furin). To solve this problem, we designed HBD deletion construct and HBD partial deletion (CKAA deletion) constructs.
HBD deletion construct 은 SOD3-WT 과 비교시, homogeneous tetramer 형태의 SOD3 생산이 가능하였다. 그러나 발현율 및 enzyme activity가 감소함을 확인하였다. Compared to SOD3-WT, the HBD deletion construct was capable of producing SOD3 in the form of a homogeneous tetramer. However, it was confirmed that the expression rate and enzyme activity decreased.
HBD partial deletion (CKAA deletion)은 SOD3-WT과 비교시, homogeneous tetramer 형태의 SOD3 생산이 가능하였고, 또한 발현율은 약2배 향상되었으며, enzyme activity는 유지되는 것을 확인하였다. 또한 R213A, R213G와 R213L mutants를 도입함으로써, 기존의 proteolytic cleavage (Furin)로 인한 HBD deletion도 억제할 수 있었다.Compared to SOD3-WT, HBD partial deletion (CKAA deletion) was able to produce SOD3 in the form of a homogeneous tetramer, and the expression rate was improved by about 2-fold, and enzyme activity was confirmed to be maintained. In addition, by introducing R213A, R213G, and R213L mutants, HBD deletion caused by existing proteolytic cleavage (Furin) could be suppressed.
실시예 2. HSA-SOD3-CKAA deletion Construction and TestExample 2. HSA-SOD3-CKAA deletion Construction and Test
1) HSA-SOD3-CKAA deletion Construction1) HSA-SOD3-CKAA deletion Construction
① HSA - partial HBD : HSA - CKAA deletion① HSA - partial HBD: HSA - CKAA deletion
; HSA(Human serum albumin); Human serum albumin (HSA)
"ASDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSAS", HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE" 를 갖는 SOD3, Linker "GGGGS", Histag "HHHHHH"를 확인할 수 있다."", SOD3 with "RKKRRRESE" , a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed, Linker "GGGGS", Histag "HHHHHH" can be confirmed.
② HSA - partial HBD - point mutation : HSA - CKAA deletion - R213G② HSA - partial HBD - point mutation: HSA - CKAA deletion - R213G
③ HSA - partial HBD - point mutation : HSA - CKAA deletion - R213A③ HSA - partial HBD - point mutation: HSA - CKAA deletion - R213A
④ HSA - partial HBD - point mutation : HSA - CKAA deletion - R213L ④ HSA - partial HBD - point mutation: HSA - CKAA deletion - R213L
SOD3-CKAA
deletion-WTHSA-
SOD3-CKAA
deletion-WT
SOD3-CKAA deletion
-R213GHSA-
SOD3-CKAA deletion
-R213G
SOD3-CKAA deletion
-R213AHSA-
SOD3-CKAA deletion
-R213A
SOD3-CKAA deletion
-R213LHSA-
SOD3-CKAA deletion
-R213L
2) HSA-SOD3-CKAA deletion Test2) HSA-SOD3-CKAA deletion Test
① Expression test ① Expression test
도 6 및 7은 HSA-SOD3-CKAA deletion의 Expression test 결과를 나타내는 그래프이다. 도 6은 SDS-PAGE 분석 그래프이고, 도 7은 단백질 발현 수율 분석 그래프이다. Figures 6 and 7 are graphs showing the expression test results of HSA-SOD3-CKAA deletion. Figure 6 is a SDS-PAGE analysis graph, and Figure 7 is a protein expression yield analysis graph.
살펴보면, HSA-SOD3-WT 에서는 high oligomerization 패턴이 확인되었다. 그러나 HSA-SOD3-HBD_deletion(HBD 완전 제거) 및 HSA-partial deletion에서는 High oligomerization이 확인되지 않았다. Upon inspection, a high oligomerization pattern was confirmed in HSA-SOD3-WT. However, high oligomerization was not confirmed in HSA-SOD3-HBD_deletion (complete HBD removal) and HSA-partial deletion.
또한 HSA-Partial deletion construction은 약 2배 이상 발현율이 향상되는 것을 확인하였다. In addition, HSA-Partial deletion construction was confirmed to improve the expression rate by more than 2 times .
② Tetramer formation test② Tetramer formation test
도 8은 HSA-SOD3-CKAA deletion의 Tetramer formation test 결과를 나타내는 그래프이다. 도 8은 SE-HPLC 분석 그래프이다. Figure 8 is a graph showing the results of the tetramer formation test of HSA-SOD3-CKAA deletion. Figure 8 is a SE-HPLC analysis graph.
살펴보면, HSA-SOD3-WT에서는 high oligomerization 패턴이 확인되었으나, HSA- SOD3-HBD_deletion 및 HSA-partial deletion에서는 High oligomerization이 확인되지 않고, homogenous tetramer가 형성되는 것을 확인하였다. Upon examination, a high oligomerization pattern was confirmed in HSA-SOD3-WT, but high oligomerization was not confirmed in HSA-SOD3-HBD_deletion and HSA-partial deletion, and a homogenous tetramer was confirmed to be formed .
③ Enzyme activity test③ Enzyme activity test
도 9 및 도 10은 HSA-SOD3-CKAA deletion의 Enzyme activity test 결과를 나타내는 그래프이다. Figures 9 and 10 are graphs showing the enzyme activity test results of HSA-SOD3-CKAA deletion.
살펴보면, HBD deletion constructs는 활성이 약 70% 감소되는 것을 확인하였다. 그러나 SOD3-CKAA-deletion와 HSA-SOD3-CKAA-deletion construct는 SOD3-WT 과 유사활성을 갖는 것을 확인하였다. Upon examination, it was confirmed that the activity of HBD deletion constructs was reduced by about 70%. However , SOD3-CKAA-deletion and HSA-SOD3-CKAA-deletion constructs were confirmed to have similar activity to SOD3-WT .
또한 HSA-SOD3-HBD deletion construct는 활성이 약 70% 감소되는 것을 확인하였다. 그러나 HSA-SOD3-CKAA deletion variants는 SOD3-WT과 유사 활성을 갖는 것을 확인하였다. 또한 HSA-SOD3-CKAA deletion variants는 HSA-SOD3-WT과 유사 활성 갖는 것을 확인하였다. Additionally, it was confirmed that the activity of the HSA-SOD3-HBD deletion construct was reduced by about 70%. However , HSA-SOD3-CKAA deletion variants were confirmed to have similar activity to SOD3-WT. In addition, HSA-SOD3-CKAA deletion variants were confirmed to have similar activity to HSA-SOD3-WT.
3) 평가 분석 고찰3) Evaluation analysis consideration
의약품 개발을 목적으로, 안정성 및 Half-life 증가를 위해 HSA(Human serum albumin) fusion HBD deletion construct와 HSA fusion HBD partial deletion(CKAA deletion) Construct를 디자인하였다. For the purpose of drug development, HSA (Human serum albumin) fusion HBD deletion construct and HSA fusion HBD partial deletion (CKAA deletion) construct were designed to increase stability and half-life.
HSA fusion HBD deletion construct은 HSA fusion SOD3-WT 과 비교시, 약 80% Homogeneous tetramer 형태의 SOD3 생산이 가능하였다. 하지만, 발현율 및 Enzyme activity가 감소되는 것을 확인하였다.Compared to HSA fusion SOD3-WT, the HSA fusion HBD deletion construct was capable of producing approximately 80% homogeneous tetramer form of SOD3. However, it was confirmed that the expression rate and enzyme activity were decreased.
그러나 HSA fusion HBD partial deletion(CKAA deletion)은 HSA fusion SOD3-WT과 비교시, Homogeneous tetramer 형태의 SOD3 생산이 가능하였고, 또한 발현율은 약 2배 향상되었 으며, Enzyme activity도 잘 유지되는 것을 확인 하였다. However , compared to HSA fusion SOD3-WT, HSA fusion HBD partial deletion (CKAA deletion) was able to produce SOD3 in the form of a homogeneous tetramer, and the expression rate was improved by about 2 times, and enzyme activity was also well maintained .
실시예 3. 재조합 SOD3 단백질의 생산 방법Example 3. Method for producing recombinant SOD3 protein
1) 상기 실시예 1에 따라, 재조합 SOD3 단백질의 생산 방법은, ① partial HBD(CKAA deletion) 형성단계 : HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"가 갖는 partial HBD SOD3를 형성하는 단계, ② partial HBD - point mutation(CKAA deletion - R213G) 형성단계 : HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK G RRESE"가 형성된 partial HBD & point mutation SOD3를 형성하는 단계, ③ partial HBD - point mutation(CKAA deletion - R213A) : HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK A RRESE"가 형성된 partial HBD & point mutation SOD3를 형성하는 단계, ④ partial HBD - point mutation(CKAA deletion - R213L) : HBD(Heparin binding domain)의 219-222AA(CKAA) 제거된 partial HBD인 "RKKRRRESE"에서 R213 mutant 처리하여 "RKK L RRESE"가 형성된 partial HBD & point mutation SOD3를 형성하는 단계를 선택적으로 포함한다.1) According to Example 1, the method for producing recombinant SOD3 protein is ① partial HBD (CKAA deletion) formation step: "RKKRRRESE" , a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed, has Step of forming partial HBD SOD3, ② Partial HBD - point mutation (CKAA deletion - R213G) formation step: Processing R213 mutant in "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed . Step of forming partial HBD & point mutation SOD3 where "RKK G RRESE" is formed, ③ partial HBD - point mutation (CKAA deletion - R213A): "RKKRRRESE", a partial HBD with 219-222AA (CKAA) of HBD (Heparin binding domain) removed "Step of forming partial HBD & point mutation SOD3 in which "RKK A RRESE" is formed by processing R213 mutant, ④ partial HBD - point mutation (CKAA deletion - R213L): 219-222AA (CKAA) of HBD (Heparin binding domain) Optionally includes the step of forming partial HBD & point mutation SOD3, in which "RKK L RRESE" is formed, by treating R213 mutant in "RKKRRRESE", which is a removed partial HBD.
2) 상기 실시예 2에 따라, 재조합 SOD3 단백질의 생산 방법은, ① HSA - partial HBD(HSA - CKAA deletion) 형성 단계, ② HSA - partial HBD - point mutation(HSA - CKAA deletion - R213G) 형성 단계, ③ HSA - partial HBD - point mutation(HSA - CKAA deletion - R213A) 형성 단계, ④ HSA - partial HBD - point mutation(HSA - CKAA deletion - R213L) 형성 단계를 선택적으로 포함한다. 2) According to Example 2, the method for producing recombinant SOD3 protein includes the following steps: ① HSA - partial HBD (HSA - CKAA deletion) formation step, ② HSA - partial HBD - point mutation (HSA - CKAA deletion - R213G) formation step, Optionally includes ③ HSA - partial HBD - point mutation (HSA - CKAA deletion - R213A) formation step, and ④ HSA - partial HBD - point mutation (HSA - CKAA deletion - R213L) formation step.
실시예 4. 재조합 SOD3 단백질의 발현 촉진 방법 Example 4. Method for promoting expression of recombinant SOD3 protein
상기 실시예 1, 2, 3에 근거한다.Based on Examples 1, 2, and 3 above.
실시예 5. 재조합 SOD3 단백질의 안정화 방법 Example 5. Method for stabilizing recombinant SOD3 protein
상기 실시예 1, 2, 3에 근거한다.Based on Examples 1, 2, and 3 above.
실시예 6. 실시예 3, 4, 5의 방법에 따라 생산된 SOD3 단백질 Example 6. SOD3 protein produced according to the method of Examples 3, 4, and 5
실시예 6는 상기 실시예 3의 생산 방법에 따라 생산된 SOD3 단백질이다. Example 6 is the SOD3 protein produced according to the production method of Example 3 above.
Claims (8)
R213G, R213A 또는 R213L 변이 중 어느 하나의 변이를 일으키는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법.In claim 1,
A method for producing a recombinant SOD3 protein, characterized in that it further comprises a step of causing any one of the R213G, R213A or R213L mutations.
HSA(Human serum albumin)를 접합하는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법.In claim 1,
A method for producing recombinant SOD3 protein, characterized in that it further comprises the step of conjugating HSA (Human serum albumin).
HSA(Human serum albumin)를 접합하는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법.In claim 2,
A method for producing recombinant SOD3 protein, characterized in that it further comprises the step of conjugating HSA (Human serum albumin).
R213G, R213A 또는 R213L 변이 중 어느 하나의 변이를 일으키는 단계를 추가로 포함하는 것을 특징으로 하는, 재조합 SOD3 단백질의 생산 방법.In claim 3,
A method for producing a recombinant SOD3 protein, characterized in that it further comprises a step of causing any one of the R213G, R213A or R213L mutations.
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