KR101658082B1 - Method for Preparing Recombinant Protein Using EDA as a Fusion Expression Partner - Google Patents
Method for Preparing Recombinant Protein Using EDA as a Fusion Expression Partner Download PDFInfo
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- KR101658082B1 KR101658082B1 KR1020150007133A KR20150007133A KR101658082B1 KR 101658082 B1 KR101658082 B1 KR 101658082B1 KR 1020150007133 A KR1020150007133 A KR 1020150007133A KR 20150007133 A KR20150007133 A KR 20150007133A KR 101658082 B1 KR101658082 B1 KR 101658082B1
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- protein
- eda
- gene
- target protein
- expression
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Abstract
본 발명은 EDA (E. coli KDPG aldolase)를 융합파트너로 이용한 재조합 단백질의 제조방법에 관한 것으로, 더욱 상세하게는 EDA 유전자를 융합파트너로 포함하는 발현벡터 또는 유전자 구조체(gene construct) 및 이를 이용한 재조합 단백질의 제조방법에 관한 것이다. 본 발명에 따른 EDA를 융합파트너로 이용한 재조합 단백질의 제조방법은 난발현 단백질의 활성 발현을 유도하고, 목적 단백질의 수용성 및 발현율을 향상시켜, 다양한 목적 단백질의 의료용 및 산업용으로 개발 및 생산하는데 유용하다.The present invention relates to a method for producing a recombinant protein using EDA ( E. coli KDPG aldolase) as a fusion partner, and more particularly, to an expression vector or gene construct comprising an EDA gene as a fusion partner, And a method for producing the protein. The method for producing a recombinant protein using EDA according to the present invention as a fusion partner is useful for inducing the expression of an egg-expressing protein and enhancing the water solubility and expression ratio of a target protein and for developing and producing various target proteins for medical use and industrial use .
Description
본 발명은 EDA (E. coli KDPG aldolase)를 융합파트너로 이용한 재조합 단백질의 제조방법에 관한 것으로, 더욱 상세하게는 EDA 유전자를 융합파트너로 포함하는 발현벡터 또는 유전자 구조체(gene construct) 및 이를 이용한 재조합 단백질의 제조방법에 관한 것이다.
The present invention relates to a method for producing a recombinant protein using EDA ( E. coli KDPG aldolase) as a fusion partner, and more particularly, to an expression vector or gene construct comprising an EDA gene as a fusion partner, And a method for producing the protein.
생명공학 기술에 의해 생산되는 단백질에는 일반적으로 면역 조절 및 효소 저해제 및 호르몬 같은 의약 및 연구용 단백질과 진단용 단백질이나 반응 첨가 효소와 같은 산업용 단백질로 대별될 수 있으며, 이 두 가지 단백질들을 중심으로 생산 공정 기술 개발 및 산업화가 추진되고 있다. 특히 재조합 미생물 기술을 이용하여 유용한 재조합 단백질을 생산할 때, 유전정보가 잘 알려져 있으며 다양한 벡터 시스템을 구축하고 있고, 비교적 값싼 배지에서 빠르게 고농도로 배양할 수 있는 장점을 갖는 대장균이 연구 또는 상업적 목적으로 다양하게 사용되고 있다.Proteins produced by biotechnology can generally be divided into medicinal and research proteins such as immunomodulatory and enzyme inhibitors and hormones and industrial proteins such as diagnostic proteins and reaction additive enzymes. Development and industrialization. Especially when producing useful recombinant proteins using recombinant microorganism technology, E. coli, which has a well-known genetic information and has a variety of vector systems, and has the advantage of rapidly growing at a high concentration in a relatively inexpensive medium, .
대장균에서 재조합 단백질을 생산함에 있어, 강력한 유도성(inducible) 프로모터를 갖춘 다양한 발현벡터가 개발되어 외래단백질의 생산에 이용되어 왔다. 그러나 숙주세포로 대장균을 이용하는 경우 제조하고자 하는 단백질이 대장균 내의 단백질 분해효소에 의해 분해되어 수율이 낮아지는 경우가 많으며, 특히 분자량이 10kDa 이하의 작은 크기의 폴리펩타이드의 발현에서 이러한 경향이 심한 것으로 알려져 있다. 뿐만 아니라 일반적으로 대장균은 단백질의 전사(transcription)와 전이(translation)가 거의 동시에 일어나기 때문에 재조합 단백질의 과다 발현시 불용성 응집체(inclusion body)를 형성하는 경우가 많으며, 응집체로 발현된 폴리펩타이드의 경우 접힘(folding) 중간체가 분자 상호간의 다이설파이드 결합(intermolecular disulfide bond) 또는 소수성 상호작용(hydrophobic interaction)에 의해 숙주세포의 다른 단백질 불순물들[샤페론(chaperon), 라이보좀(ribosome), 초기인자 등]과 비선택적으로 결합함으로써 목적 폴리펩타이드의 응집체 내 순도가 떨어지는 단점이 있다. 또한 이렇게 발현된 단백질을 활성형으로 만들기 위해서는 구아니딘-하이드로클로라이드(Guanidine hychloride)나 우레아(urea) 같은 변성체를 사용하여 용해시킨 후 희석하는 재접힘(refolding) 과정을 거쳐야 하는데 이때 단백질이 활성형으로 접히지 않는 등 생산 수율이 감소하는 문제점이 있다 (Marston FA et al., Biochem J 240(1):1-12, 1986).In the production of recombinant proteins in E. coli, various expression vectors with a strong inducible promoter have been developed and used in the production of foreign proteins. However, when Escherichia coli is used as a host cell, the protein to be produced is often degraded by proteolytic enzymes in E. coli, resulting in a low yield. Especially, it is known that this tendency is severe in the expression of small-sized polypeptides having a molecular weight of 10 kDa or less have. In addition, Escherichia coli generally produces insoluble aggregates in the over-expression of recombinant proteins because transcription and translation of the proteins occur at almost the same time. In the case of polypeptides expressed as aggregates, folding intermediates can interact with other protein impurities in the host cell (chaperon, ribosome, initial factor, etc.) by intermolecular disulfide bonds or hydrophobic interactions There is a drawback that the purity in the aggregate of the desired polypeptide is lowered by non-selective binding. In order to make the expressed protein active, it must be refolded by diluting with a modified product such as guanidine hydrochloride (urea) or urea (urea) (Marston FA et al., Biochem J 240 (1): 1-12, 1986).
대장균 내에서 활성형의 재조합 단백질을 고수율로 얻기 위해 대장균의 단백질 발현 속도를 늦추어 목적 단백질의 수용도를 높여주는 저온 발현방법(Hammarstrom et al., Protein Sci. 11:313-321, 2002), mRNA의 친화력을 높힐 수 있는 진보된 형태의 프로모터를 사용하거나 유도(induction) 조건의 최적화하는 방법 (Qing et al., Nat Biotechnol. 22:877-882, 2004) 및 분자 샤페론 또는 단백질 접힘 조절자와 목적 단백질을 동시 발현시키는 방법(de Marco & De Marco, J Biotechnol. 109:45-52, 2004) 등이 시도되고 있지만, 목적 단백질의 아미노 말단에 융합파트너를 융합시켜서 발현시키는 방법이 가장 일반적으로 사용되고 있다. 융합파트너를 융합시켜 발현할 경우 목적 단백질을 수용성으로 유도할 수 있는 장점이 있으며, 또한 엔테로카이네이즈(enterokinase)와 같은 효소를 이용하여 융합파트너를 제거함으로써 아미노 말단의 메티오닌 문제를 해결할 수 있다.(Hammarstrom et al., Protein Sci. 11: 313-321, 2002), which increases the acceptability of the target protein by slowing the protein expression rate of Escherichia coli to obtain an active recombinant protein in E. coli in high yield, (Qing et al., Nat Biotechnol. 22: 877-882, 2004) and molecular chaperones or protein folding modulators and the like, by using an advanced form of promoter capable of enhancing the affinity of the mRNA or by optimizing the induction conditions A method of simultaneously expressing a target protein (de Marco & De Marco, J Biotechnol. 109: 45-52, 2004) has been attempted. However, a method in which a fusion partner is fused to the amino terminus of a target protein and expressed is most commonly used have. When the fusion partner is fused and expressed, the target protein can be induced to be soluble. Further, the methionine problem at the amino terminal can be solved by removing the fusion partner using an enzyme such as enterokinase.
실제로 대장균 내에서 외래단백질의 수용성 높은 발현을 유도하는 여러 융합파트너가 지난 수년 동안 연구되고 보고되어왔다 (Esposito & Chatterjee, Curr Opin Biotechnol. 17:353-358 2006; Kapust & Waugh, Protein Sci. 8:1668-1674, 1999; Sachdev & Chirgwin, Biochem. Biophys. Res. Commun. 244:933-937, 1998). 가장 많이 연구된 대표적인 융합파트너로는 말토오즈 결합 단백질 (Maltose binding protein; MBP), 글루타치온-S-전이효소(Glutathione-S-transferase; GST), 티오레독신 (Thioredoxin; Trx), NusA 등이 있다. 말토오스 결합 단백질의 경우 이량체 형성에 관여하는 부분에 소수성을 띠는 아미노산이 모여 만들어진 넓은 소수성 틈새가 새로 합성되는 단백질의 소수성 부위를 효과적으로 감춰주어 목적단백질이 불용성 응집체가 되는 것을 방지해 주며, 티오레독신은 목적단백질의 다이설파이드 결합을 도와주고, NusA의 경우 대장균에서 과량으로 발현되었을 때 활성형으로 접히는 능력이 매우 뛰어나므로 뒤따라 발현되는 목적단백질의 올바른 접힘을 유도한다고 알려져 있다(Bach H et al., J Mol Biol 312:79-93, 2001; Edward RL et al.,Nat Biotechnol 11:187-193, 1993; Davis GD et al., Biotechnol Bioeng 65:382-388, 1999).In fact, several fusion partners have been studied and reported over the past several years to induce high expression of foreign proteins in E. coli (Esposito & Chatterjee, Curr Opin Biotechnol. 17: 353-358 2006; Kapust & Waugh, Protein Sci. 8: Sachdev & Chirgwin, Biochem. Biophys. Res. Commun. 244: 933-937, 1998). Maltose binding protein (MBP), Glutathione-S-transferase (GST), Thioredoxin (Trx), NusA, and the like are among the most studied fusion partners . In the case of the maltose binding protein, a wide hydrophobic gap formed by the amino acids having hydrophobicity in the portion involved in dimer formation effectively hides the hydrophobic region of the newly synthesized protein and prevents the target protein from becoming an insoluble aggregate. Bacillus is known to help the disulfide bond of the target protein, and NusA is highly active in folding when expressed in E. coli, leading to proper folding of the expressed target protein (Bach H et al. , J Mol Biol 312: 79-93, 2001; Edward RL et al., Nat Biotechnol 11: 187-193, 1993; Davis GD et al., Biotechnol Bioen 65: 382-388, 1999).
이러한 융합파트너는 친화 크로마토그래피(affinity chromatography) 방법을 이용하여 융합발현 된 목적단백질을 쉽게 정제할 수 있다는 장점이 있으며, 각기 다른 분자생물학적 특성을 이용하여 목적단백질의 접힘을 돕는다고 알려져 있다. 하지만 이러한 융합파트너들은 목적단백질에 비해 상대적으로 크기가 크기 때문에 융합 부분의 크기에 따라 목적단백질의 수율이 현저히 떨어진다는 단점과 의료목적 또는 산업적으로 유용한 단백질들에 모두 범용적으로 작용하지 않는다는 단점이 있다. 또한, 이합체(dimer)를 형성하여 목적 단백질 또한 이합체(dimer)형태로 생산되는 경우도 있을뿐 아니라, 목적단백질을 수용성으로 발현을 유도하더라도 고유한 기능을 수행하는 활성형으로 발현을 유도하는 데는 실패하는 경우가 많고, 적합한 용도로 이용되기 위하여 융합파트너의 제거 과정이 추가되어야 하는 공정상의 불합리성을 지닌다는 문제점이 있다. Such a fusion partner has an advantage that it can easily purify a target protein expressed by fusion using an affinity chromatography method, and it is known that it helps folding of a target protein by using different molecular biological characteristics. However, these fusion partners have a disadvantage in that the yield of the target protein is remarkably lowered depending on the size of the fusion region, and the protein is not universally used for medical purposes or industrially useful proteins because it is relatively large in size compared to the target protein . In addition, there are cases in which a target protein is also produced in the form of a dimer by forming a dimer, and it is difficult to induce expression in an active form that performs a unique function even if the target protein is expressed in a water- There is a problem in that there is a process irrationality in which the removal process of the fusion partner must be added in order to be used for a suitable application.
지금까지 생명공학 기술에 의해 산업적으로 생산된 국내의 재조합 단백질은 발현 가능한 재조합 단백질들의 생산 공정개발에 치중되어 있어서 핵심 원천 기술인 발현시스템 개발은 외국에 비해 상대적으로 모방 또는 개량 수준의 부가기술로서 개발되고 있다. 또한, 상업적으로나 의약적으로 매우 중요한 단백질은 분비 단백질(secretory protein) 및 막 단백질(membrane protein)들로서 대장균 내에서 발현 시 불용성 응집체를 만드는 난발현성 단백질(difficult-to-express proteins)이여서 개발이 지연되고 있다. 이를 극복하기 위해서는 다양한 장점을 지니고 있는 대장균 시스템을 활용하여 난발현 단백질의 발현효율을 증대시키기 위한 발현시스템을 개발하여, 이에 관한 원천 기반기술을 확보하는 것이 중요하다. 또한, 대장균에서 재조합 단백질의 과다 발현은 외부 환경으로부터 받는 스트레스(열 충격, 아미노산 고갈 등)와 비슷한 영향을 준다고 알려진바 있다(Hoffman F & Rinas U, Adv Biochem Eng Biotechnol, 89:73-92, 2004).So far, domestic recombinant proteins produced industrially by biotechnology are focused on the development of production process of recombinant proteins that can be expressed, so development of expression system, which is a core source technology, is developed as an additional technology of imitation or improvement level relative to foreign countries have. In addition, proteins that are commercially or medically very important are secretory proteins and membrane proteins, which are difficult-to-express proteins that produce insoluble aggregates when expressed in E. coli, have. In order to overcome this problem, it is important to develop an expression system for enhancing the expression efficiency of egg-expressing proteins by utilizing an E. coli system having various advantages, and to secure a source-based technology related thereto. In addition, overexpression of recombinant proteins in E. coli has been known to have effects similar to the stresses (heat shock, amino acid depletion, etc.) from the external environment (Hoffman F & Rinas U, Adv Biochem Eng Biotechnol, 89: 73-92, 2004 ).
이에, 본 발명자들은 대장균에 단백질의 올바른 접힘을 저해하는 스트레스를 주어 과발현되는 단백질을 범용성 융합파트너로 이용한 재조합 단백질 발현 시스템을 구축하고자 예의 노력한 결과, EDA를 융합파트너로 이용하여 목적단백질과 융합 발현시킬 경우, 재조합 단백질의 수용성 발현율이 현저히 향상되는 것을 확인하고 본 발명을 완성하였다.
Thus, the present inventors have made efforts to establish a recombinant protein expression system using overexpressed proteins as a universal fusion partner by giving stress to inhibit the correct folding of the protein in E. coli. As a result, it has been found that EDA is used as a fusion partner, , The water-soluble expression rate of the recombinant protein was remarkably improved, and the present invention was completed.
본 발명의 목적은 EDA 유전자를 융합파트너로 이용하여 목적 단백질의 수용성 및 접힘을 향상시킬 수 있는, 재조합 단백질 생산용 발현벡터 또는 유전자 구조체(gene construct)를 제공하는데 있다.It is an object of the present invention to provide an expression vector or gene construct for recombinant protein production which can improve the water solubility and folding of a target protein by using EDA gene as a fusion partner.
본 발명의 다른 목적은 상기 발현벡터로 형질전환 되거나 또는 유전자 구조체(gene construct)가 삽입된 재조합 미생물 및 이를 이용한 재조합 단백질의 제조방법을 제공하는데 있다.
It is another object of the present invention to provide a recombinant microorganism transformed with the above expression vector or a gene construct inserted therein and a method for producing a recombinant protein using the recombinant microorganism.
상기 목적을 달성하기 위하여, 본 발명은 목적 단백질을 코딩하는 유전자와 서열번호 34로 표시되는 EDA 유전자가 연결되어 있는 유전자 구조체(gene construct)를 제공한다.In order to achieve the above object, the present invention provides a gene construct in which a gene encoding a target protein and an EDA gene represented by SEQ ID NO: 34 are linked.
본 발명은 또한, 목적 단백질 유전자와 서열번호 34로 표시되는 EDA 유전자를 융합파트너로 포함하는 목적 단백질 생산용 발현벡터를 제공한다.The present invention also provides an expression vector for producing a target protein comprising a target protein gene and an EDA gene represented by SEQ ID NO: 34 as fusion partners.
본 발명은 또한, 상기 유전자 구조체(gene construct) 또는 상기 발현벡터가 도입되어 있는 재조합 미생물을 제공한다.The present invention also provides a recombinant microorganism into which said gene construct or said expression vector has been introduced.
본 발명은 또한, 상기 재조합 미생물을 배양하여 목적 단백질의 발현을 유도한 다음, 이를 회수하는 단계를 포함하는 재조합 단백질의 제조방법을 제공한다.The present invention also provides a method for producing a recombinant protein comprising culturing the recombinant microorganism to induce expression of a target protein, and recovering the recombinant protein.
본 발명은 또한, 상기 방법에 의해 제조되고, EDA와 목적 단백질이 융합된 재조합 단백질을 제공한다.
The present invention also provides a recombinant protein produced by the above method, wherein the EDA and the target protein are fused.
본 발명에 따른 EDA를 융합파트너로 이용한 재조합 단백질의 제조방법은 난발현 단백질의 활성 발현을 유도하고, 목적 단백질의 수용성 및 발현율을 향상시켜, 다양한 목적 단백질의 의료용 및 산업용으로 개발 및 생산하는데 유용하다.
The method for producing a recombinant protein using EDA according to the present invention as a fusion partner is useful for inducing the expression of an egg-expressing protein and enhancing the water solubility and expression ratio of a target protein and for developing and producing various target proteins for medical use and industrial use .
도 1은 목적 단백질 유전자만을 단독 포함하는 발현벡터(A), EDA 유전자와 목적 단백질 유전자의 융합 발현벡터(B) 및 EDA 유전자와 목적 단백질 유전자 사이에 엔테로키나제 인식부위를 코딩하는 폴리뉴클레오티드가 연결된 융합 발현벡터(C)의 도식도이다.
도 2는 7 종류의 목적 단백질(A: hG-CSF, B: hFTN-L, C: ppGRN, D: mpINS, E: IL2, F: EGF, G: ADI)과 2 종류의 융합파트너(EDA, GST)와의 융합발현 및 단독발현 결과의 SDS-PAGE 분석을 나타낸 것이다.
도 3은 (A) 7 종류 목적 단백질의 단독발현과 EDA 융합발현과의 수용성 비교, (B) 37℃ 배양조건에서 EDA 융합발현과 GST 융합발현과의 수용성 비교 및 (C) 20℃ 배양조건에서 EDA 융합발현과 GST 융합발현과의 수용성 비교한 결과이다.
도 4는 EDA와 융합발현된 아르기닌 탈이민효소(ADI)의 활성도 비교 결과이다 (A: SDS-PAGE 분석, B: 활성도).
도 5는 EDA와 융합발현된 hG-CSF와 EDA가 제거된 hG-CSF의 SDS-PAGE (A) 및 웨스턴블롯 (B) 결과이다. (C)는 상업적으로 판매하는 hG-CSF와 EDA가 제거된 hG-CSF의 원편광 이색성 분광(CD) 분석 그래프 결과이다.
도 6 (A)는 EDA와 융합발현된 hFTN-L과 EDA가 제거된 hFTN-L의 SDS-PAGE 분석 결과이며, EDA와 융합발현된 hFTN-L (B) 및 EDA가 제거된 hFTN-L (C)의 투과전자현미경(TEM) 사진을 나타낸 것이다.Brief Description of the Drawings Fig. 1 is a schematic diagram showing an embodiment of the present invention. Fig. 1 is a schematic diagram of a fusion protein comprising an expression vector (A) containing only a desired protein gene, a fusion expression vector (B) of EDA gene and a desired protein gene, (C). ≪ / RTI >
FIG. 2 is a graph showing the relationship between the number of target proteins (A: hG-CSF, B: hFTN-L, C: ppGRN, D: mpINS, E: IL2, F: EGF, G: ADI) GST) and SDS-PAGE analysis of the results of single expression.
FIG. 3 shows (A) the solubilization of 7 kinds of target proteins and the acceptability of EDA fusion expression, (B) the water solubility comparison of EDA fusion expression with GST fusion expression at 37 ° C culture condition, and (C) EDA fusion expression and GST fusion expression.
FIG. 4 shows the result of comparing the activity of arginine deaminase (ADI) fused with EDA (A: SDS-PAGE analysis, B: activity).
FIG. 5 shows results of SDS-PAGE (A) and Western blot (B) of hG-CSF in which hG-CSF and EDA fused with EDA were removed. (C) is the result of a circularly polarized dichroism spectroscopy (CD) analysis of commercially available hG-CSF and EDA-free hG-CSF.
FIG. 6 (A) shows the results of SDS-PAGE analysis of hFTN-L and EDA-deleted hFTN-L fused with EDA and hFTN-L (B) C (TEM) photographs.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
대장균에서 목적 단백질은 불용성 응집체로 발현되는 경우가 많다. 즉, 대장균 발현시스템 내에서는 전사와 전이가 거의 동시에 일어나며, 재조합 단백질의 과다 발현 시, 목적 단백질 또는 대장균 숙주세포 내 다른 단백질들의 접힘(folding) 중간체들 사이의 비특이적인 결합에 의해 인접한 하이드로포빅(hydrophobic) 폴리펩타이드 체인들은 응집체(inclusion body)로 알려진 불용성 응집체를 형성한다고 알려져 있다. 단백질의 접힘을 저해하는 조건에서도 활성이 유지되며 발현량이 우수한 단백질은 단백질의 잘못된 접힘(misfolding)을 극복하기 위한 대장균의 생체 메커니즘에 관여할 수 있고, 단백질 구조가 안정적이며 자체 접힘 능력이 뛰어나므로 융합파트너로서 효과적으로 이용될 수 있다. 대장균 융합발현파트너 중에서 가장 효율성이 높다고 알려진 융합발현파트너는 말토오즈 결합 단백질(Maltose binding protein, 'MBP'; Bedouelle and Duplay, Eur J Biochem, 1998) 이다. 하지만 상당수의 단백질에 대해서 효과적이라고 알려진 MBP도 많은 단백질에 대해서 수용성 발현 개선 효과를 보이지 못하고 있다 (Hammarstrom et al., J Struct Funct Genomics, 2006).In E. coli, the target protein is often expressed as an insoluble aggregate. That is, in the E. coli expression system, transcription and metastasis occur almost simultaneously, and during overexpression of the recombinant protein, the hydrophobic interaction between the target protein or the folding intermediates of other proteins in the E. coli host cell ) Polypeptide chains are known to form insoluble aggregates known as inclusion bodies. The protein retains its activity even under the conditions of inhibiting the folding of the protein, and the protein having the excellent expression amount can participate in the biomechanism of E. coli to overcome the misfolding of the protein, and the protein structure is stable and self-folding ability is excellent. It can be effectively used as a partner. Maltose binding protein (MBP) (Bedouelle and Duplay, Eur J Biochem , 1998) is the most effective fusion partner of Escherichia coli fusion expression partners. However, MBP, which is known to be effective against a large number of proteins, does not exhibit an improved water-soluble expression for many proteins (Hammarstrom et al., J Struct Funct Genomics, 2006).
본 발명에서는 EDA 유전자를 융합파트너로 이용하여 다양한 목적 단백질을 생산함으로써, 범용적으로 적용 가능한 미생물 기반 단백질 활성 발현 시스템을 구축하였다. In the present invention, a variety of target proteins are produced by using the EDA gene as a fusion partner, thereby constructing a universally applicable microbial-based protein activity expression system.
본 발명에서는 목적 단백질의 수용성 발현 및 올바른 접힘(folding)을 유도하기 위하여 EDA를 융합파트너로 사용하였고, 그 결과 재조합 단백질의 수용성 발현이 현저히 향상되었음을 규명하였다.In the present invention, EDA was used as a fusion partner to induce water-soluble expression and proper folding of a target protein. As a result, water-soluble expression of the recombinant protein was remarkably improved.
따라서, 본 발명은 일 관점에서, 목적 단백질을 코딩하는 유전자와 서열번호 34로 표시되는 EDA 유전자가 연결되어 있는 유전자 구조체(gene construct)에 관한 것이다. Thus, in one aspect, the present invention relates to a gene construct in which a gene encoding a target protein and an EDA gene represented by SEQ ID NO: 34 are linked.
본 발명에 있어서, 상기 목적 단백질 유전자와 상기 EDA 유전자 사이에 단백질 절단 효소 인식부위를 코딩하는 폴리뉴클레오티드가 연결되어 있는 것을 특징으로 할 수 있다.In the present invention, a polynucleotide encoding a protein cleavage enzyme recognition site may be connected between the target protein gene and the EDA gene.
또한, 상기 목적 단백질 유전자와 상기 EDA 유전자는 분리정제용 태그를 코딩하는 폴리뉴클레오티드와 작동 가능하도록 연결되어 있는 것을 특징으로 할 수 있다.In addition, the target protein gene and the EDA gene may be operatively linked to a polynucleotide encoding a tag for separation and purification.
발명은 다른 관점에서, 목적 단백질 유전자와 서열번호 34로 표시되는 EDA 유전자를 융합파트너로 포함하는 목적 단백질 생산용 발현벡터에 관한 것이다.In another aspect, the present invention relates to an expression vector for producing a target protein comprising a target protein gene and an EDA gene represented by SEQ ID NO: 34 as fusion partners.
본 발명에 있어서, 상기 목적 단백질 유전자와 융합파트너로 EDA 유전자가 연결되어 있는 것을 특징으로 할 수 있다. 본 발명의 발현벡터는 상기 융합파트너 유전자와 목적 단백질의 유전자를 일련의 순서로 포함함으로써 상기 융합파트너와 목적 단백질을 재조합 단백질로 발현할 수 있다. In the present invention, the EDA gene may be linked to the target protein gene as a fusion partner. The expression vector of the present invention can express the fusion partner and the target protein as a recombinant protein by including the fusion partner gene and the gene of the target protein in a sequential order.
본 발명에 있어서, 상기 목적 단백질 유전자와 융합파트너로 EDA 유전자 사이에 단백질 절단 효소 인식부위를 코딩하는 폴리뉴클레오티드가 연결되어 있는 것을 특징으로 할 수 있다.In the present invention, a polynucleotide encoding a protein cleavage enzyme recognition site may be connected between the target protein gene and the EDA gene as a fusion partner.
또한, 상기 목적 단백질 유전자와 융합파트너로서의 EDA 유전자는 분리정제용 태그를 코딩하는 폴리뉴클레오티드와 작동 가능하도록 연결되어 있는 것을 특징으로 할 수 있다. In addition, the EDA gene as a fusion partner with the target protein gene may be operatively linked to a polynucleotide encoding a tag for separation and purification.
본원에서, "벡터(vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 "플라스미드(plasmid)" 및 "벡터(vector)"는 때로 상호 교환적으로 사용된다. 본 발명의 목적상, 플라스미드 벡터를 이용하는 게 바람직하다. 이러한 목적에 사용될 수 있는 전형적인 플라스미드 벡터는 (a) 숙주세포당 수 개에서 수백 개의 플라스미드 벡터를 포함하도록 복제가 효율적으로 이루어지도록 하는 복제 개시점, (b) 플라스미드 벡터로 형질전환된 숙주세포가 선발될 수 있도록 하는 항생제 내성 유전자 및 (c) 외래 DNA 절편이 삽입될 수 있는 제한효소 절단부위를 포함하는 구조를 지니고 있다. 적절한 제한효소 절단 부위가 존재하지 않을지라도, 통상의 방법에 따른 합성 올리고뉴클레오타이드 어댑터(oligonucleotide adaptor) 또는 링커(linker)를 사용하면 벡터와 외래 DNA를 용이하게 라이게이션(ligation)할 수 있다. 라이게이션 후에, 벡터는 적절한 숙주세포로 형질전환되어야 한다. 형질전환은 칼슘 클로라이드 방법 또는 전기천공법(electroporation) (Neumann, et al., EMBO J., 1:841, 1982) 등을 사용해서 용이하게 달성될 수 있다.As used herein, "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several to several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector, (C) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA. After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using a calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982).
본 발명에 따른 유전자의 과발현을 위하여 사용되는 벡터는 당업계에 공지된 발현벡터가 사용될 수 있다. 본 발명의 방법에서 사용될 수 있는 뼈대 벡터는 특별히 이에 제한되는 것은 아니나, pT7, pET/Rb, pGEX, pET28a, pET-22b(+) 및 pGEX로 이루어진 군으로부터 선택되는 대장균에 형질전환 가능한 다양한 벡터를 사용할 수 있다.As a vector used for overexpression of the gene according to the present invention, an expression vector known in the art can be used. The framework vectors that may be used in the methods of the present invention include, but are not limited to, various vectors capable of transforming into E. coli selected from the group consisting of pT7, pET / Rb, pGEX, pET28a, pET-22b Can be used.
염기서열은 다른 핵산 서열과 기능적 관계로 배치될 때 "작동가능하게 연결(operably linked)" 된다. 이것은 적절한 분자(예를 들면, 전사 활성화 단백질)가 조절 서열(들)에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 조절 서열(들)일 수 있다. 예를 들면, 전서열(pre-sequence) 또는 분비 리더 (leader)에 대한 DNA는 폴리펩타이드의 분비에 참여하는 전단백질로서 발현되는 경우 폴리펩타이드에 대한 DNA에 작동가능하게 연결되고 프로모터 또는 인핸서는 서열의 전사에 영향을 끼치는 경우 코딩서열에 작동가능하게 연결되거나 또는 리보좀 결합 부위는 서열의 전사에 영향을 끼치는 경우 코딩 서열에 작동가능하게 연결되거나 또는 리보좀 결합 부위는 번역을 용이하게 하도록 배치되는 경우 코딩 서열에 작동가능하게 연결된다. 일반적으로, "작동가능하게 연결된"은 연결된 DNA 서열이 접촉하고, 또한 분비 리더의 경우 접촉하고 리딩 프레임 내에 존재하는 것을 의미한다. 그러나, 인핸서(enhancer)는 접촉할 필요가 없다. 이들 서열의 연결은 편리한 제한 효소 부위에서 라이게이션(연결)에 의해 수행된다. 그러한 부위가 존재하지 않는 경우, 통상의 방법에 따른 합성 올리고뉴클레오티드 어댑터(oligonucleotide adaptor) 또는 링커(linker)를 사용한다.A nucleotide sequence is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide, and the promoter or enhancer is a sequence Or the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site is arranged to facilitate translation, Lt; / RTI > sequence. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.
당업계에 주지된 바와 같이, 숙주세포에서 형질전환 유전자의 발현 수준을 높이기 위해서는, 해당 유전자가 선택된 발현 숙주 내에서 기능을 발휘하는 전사 및 해독 발현 조절 서열에 작동가능하도록 연결되어야만 한다. 바람직하게는 발현 조절서열 및 해당 유전자는 세균 선택 마커 및 복제 개시점(replication origin)을 같이 포함하고 있는 하나의 재조합벡터 내에 포함되게 된다. 숙주세포가 진핵세포인 경우에는, 재조합벡터는 진핵 발현 숙주 내에서 유용한 발현 마커를 더 포함하여야만 한다.As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host. Preferably the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector should further comprise a useful expression marker in the eukaryotic expression host.
상술한 재조합 벡터에 의해 형질전환된 숙주 세포는 본 발명의 또 다른 측면을 구성한다. 본원 명세서에 사용된 용어 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체 외 인자로서 또는 염색체 통합완성에 의해 복제 가능하게 되는 것을 의미한다.The host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term "transformation" means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.
물론 모든 벡터가 본 발명의 DNA 서열을 발현하는데 모두 동등하게 기능을 발휘하지는 않는다는 것을 이해하여야만 한다. 마찬가지로 모든 숙주가 동일한 발현 시스템에 대해 동일하게 기능을 발휘하지는 않는다. 그러나, 당업자라면 과도한 실험적 부담 없이 본 발명의 범위를 벗어나지 않는 채로 여러 벡터, 발현 조절 서열 및 숙주 중에서 적절한 선택을 할 수 있다. 예를 들어, 벡터를 선택함에 있어서는 숙주를 고려하여야 하는데, 이는 벡터가 그 안에서 복제되어야만 하기 때문이다. 벡터의 복제 수, 복제 수를 조절할 수 있는 능력 및 당해 벡터에 의해 코딩되는 다른 단백질, 예를 들어 항생제 마커의 발현도 또한 고려되어야만 한다.Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.
따라서, 본 발명은 또 다른 관점에서, 상기 목적 단백질을 코딩하는 유전자와 서열번호 34로 표시되는 EDA 유전자가 연결되어 있는 유전자 구조체(gene construct) 또는 상기 목적 단백질 유전자와 서열번호 34로 표시되는 EDA 유전자를 융합파트너로 포함하는 목적 단백질 생산용 발현벡터가 도입되어 있는 재조합 미생물에 관한 것이다. Therefore, in another aspect, the present invention provides a gene construct comprising a gene encoding the target protein and an EDA gene represented by SEQ ID NO: 34, or an EDA gene represented by SEQ ID NO: 34 As a fusion partner, into a recombinant microorganism into which an expression vector for production of a desired protein has been introduced.
본 발명에 있어서, 상기 유전자 구조체(gene construct)는 숙주세포의 염색체 내에 삽입되어 있는 것을 특징으로 할 수 있다.In the present invention, the gene construct may be inserted into the chromosome of the host cell.
발명이 속하는 기술분야의 당업자에게 있어 상기 유전자를 숙주세포의 게놈 염색체에 삽입하여서도 상기와 같이 재조합 벡터를 숙주세포에 도입한 경우와 동일한 효과를 가질 것은 자명하다 할 것이다.It will be apparent to those skilled in the art that the gene has the same effect as that of introducing the recombinant vector into the host cell by inserting the gene into the genome chromosome of the host cell.
본 발명에서 상기 유전자를 숙주세포의 염색체상에 삽입하는 방법으로는 통상적으로 알려진 유전자조작방법을 사용할 수 있으며, 일 예로는 레트로바이러스 벡터, 아데노바이러스 벡터, 아데노-연관 바이러스 벡터, 헤르페스 심플렉스 바이러스 벡터, 폭스바이러스 벡터, 렌티바이러스 벡터 또는 비바이러스성 벡터를 이용하는 방법을 들 수 있다.In the present invention, the gene may be inserted into the chromosome of the host cell by a commonly known gene manipulation method. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector , A poxvirus vector, a lentiviral vector, or a nonviral vector.
융합파트너와 융합할 목적 단백질이 산업용 단백질일 경우, 융합파트너가 목적 단백질의 기능을 저하할 수도 있으며, 의학용 단백질일 경우는 항원항체 반응을 야기할 수 있으므로 융합파트너는 제거되는 것이 바람직하다. 이때, 상기 단백질 절단효소 인식부위는 Xa 인자 인식부위, 엔테로키나제 인식부위, 제네나제(Genenase) I 인식 부위 또는 퓨린(Furin) 인식부위가 단독으로 사용되거나 어느 두 개 이상을 순차적으로 연결하여 사용할 수 있다.When the objective protein to be fused with the fusion partner is an industrial protein, the fusion partner may degrade the function of the target protein, and in the case of a medical protein, the fusion partner is preferably removed since it may cause an antigen-antibody reaction. Herein, the protein cleavage enzyme recognition site may be a Xa factor recognition site, an enterokinase recognition site, a Genenase I recognition site, or a purine recognition site, or two or more of them may be used in sequence have.
또한, 재조합 단백질의 분리 정제가 용이하도록, 본 발명의 벡터의 융합파트너 또는 목적 단백질의 유전자에 분리정제용 태그를 코딩하는 폴리뉴클레오티드를 작동 가능하도록 연결할 수 있다. 이때, 상기 분리정제용 태그는 GST, poly-Arg, FLAG, poly-His 및 c-myc 등이 단독으로 사용되거나 어느 두 개 이상을 순차적으로 연결하여 사용할 수 있다. 발명의 일 실시예에서, 본 발명의 벡터는 pT7 뼈대 벡터에 분리정제용 태그, 상기 융합파트너의 유전자 및 목적 단백질의 유전자를 일련의 순서로 포함함으로써 분리정제용 태그, 상기 융합파트너 및 목적 단백질을 포함하는 재조합 단백질을 발현할 수 있다.In addition, a polynucleotide encoding a tag for separation purification can be operably linked to the fusion partner of the vector of the present invention or the gene of the target protein, so that the recombinant protein can be easily separated and purified. At this time, GST, poly-Arg, FLAG, poly-His and c-myc may be used singly or two or more of them may be sequentially connected. In one embodiment of the present invention, the vector of the present invention comprises a tag for separation and purification, a gene for the fusion partner, and a gene of a desired protein in a sequence in the pT7 skeletal vector, Lt; RTI ID = 0.0 > recombinant < / RTI >
상기 목적 단백질의 유전자는 제한효소 부위를 통해 클로닝 될 수 있고, 단백질 절단효소 인식부위를 코딩하는 폴리뉴클레오티드가 사용된 경우에는 상기 폴리뉴클레오티드와 틀이 맞도록(in frame) 연결되어, 목적 단백질 분비 후 단백질 절단효소로 절단하여, 원래 형태의 목적 단백질을 생산할 수 있도록 할 수 있다.The gene of the target protein can be cloned through a restriction enzyme site, and when a polynucleotide encoding a protein cleavage enzyme recognition site is used, it is linked in frame with the polynucleotide, It can be cleaved with a protein cleaving enzyme to produce the original protein of interest.
본원의 용어, "목적 단백질(target protein)" 또는 "외래 단백질(heterologous protein)"은 업자가 대량으로 생산하고자 하는 단백질로서, 재조합 발현벡터에 상기 단백질을 코딩하는 폴리뉴클레오티드를 삽입하여 형질전환체에서 발현이 가능한 모든 단백질을 의미한다. As used herein, the term "target protein" or "heterologous protein" refers to a protein that a manufacturer intends to produce in large quantities. In the present invention, a polynucleotide encoding the protein is inserted into a recombinant expression vector, Means all proteins capable of expression.
또한, 본 발명에서, "재조합 융합 단백질"이란, 목적단백질과 EDA가 융합되어 있는 형태의 단백질을 의미한다. 보다 구체적으로, 본 발명은 당업자가 원하는 모든 단백질에 적용 가능하며, 특히, 의료, 연구용 및 산업용 단백질, 예를 들어, 항원, 항체, 세포수용체, 효소, 구조 단백질, 혈청, 세포 단백질로 이루어진 군으로부터 선택되는 생물학적 활성을 갖는 다양한 목적 단백질을 재조합 단백질 형태로 발현할 수 있다.In the present invention, the term "recombinant fusion protein" means a protein in which the target protein and EDA are fused. More specifically, the present invention is applicable to all proteins desired by a person skilled in the art, and is particularly applicable to all types of proteins, including medical, research and industrial proteins such as antigens, antibodies, cell receptors, enzymes, structural proteins, Various target proteins having the selected biological activity can be expressed in recombinant protein form.
본 발명에서는 EDA가 목적 단백질의 접힘을 돕는 융합파트너로서의 가능성을 검증하기 위하여, 대장균에서 융합파트너 없이 단독으로 발현하였을 때 불용성 응집체를 형성한다고 알려진 7개의 단백질과 융합 발현시켰다. 즉, 의료, 연구용 및 산업용 단백질로는 대표적으로 인간 프리프로-그렐린 (human prepro-ghrelin, 이하 'ppGRN'), 인간 인터루킨-2(human interleukin-2, 이하 'IL-2'), 인간 미니프로인슐린 (human minipro-insulin, 이하 'mp-INS'), 인간 상피세포 성장인자 (human epidermal growth factor, 이하 'EGF'), 인간 페리틴 가벼운 사슬(human ferritin light chain, 이하 'hFTN-L'), 인간 과립구 집락-자극인자(human granulocyte colony-stimulating factor, 이하 'G-CSF') 및 아르기닌 디이미나아제(Arginine deiminase, 이하 'ADI')를 목적 단백질로 선정하여 각각 EDA의 카르복실 말단에 삽입한 발현벡터를 제조한 후(도 1), 이를 대장균에 형질전환시키고 이로부터 재조합 단백질 형태로 생산하였다.In the present invention, in order to test the possibility of EDA as a fusion partner to help folding of the target protein, fusion proteins were expressed with seven proteins known to form insoluble aggregates when expressed alone in E. coli without a fusion partner. Examples of proteins for medical, research and industrial use include human prepro-ghrelin (hereinafter referred to as "ppGRN"), human interleukin-2 (hereinafter referred to as "IL-2" Human epidermal growth factor (hereinafter referred to as 'EGF'), human ferritin light chain (hereinafter referred to as 'hFTN-L'), human insulin, Human granulocyte colony-stimulating factor (G-CSF) and arginine deiminase (ADI) were selected as target proteins and inserted into the carboxyl terminal of EDA, respectively. After the expression vector was prepared (Fig. 1), it was transformed into Escherichia coli and the recombinant protein was produced therefrom.
따라서, 본 발명은 또 다른 관점에서, 상기 목적 단백질을 코딩하는 유전자와 서열번호 34로 표시되는 EDA 유전자가 연결되어 있는 유전자 구조체(gene construct) 또는 상기 목적 단백질 유전자와 서열번호 34로 표시되는 EDA 유전자를 융합파트너로 포함하는 목적 단백질 생산용 발현벡터가 도입되어 있는 재조합 미생물을 배양하여 재조합 단백질의 발현을 유도한 다음, 이를 회수하는 단계를 포함하는 목적 단백질의 제조방법에 관한 것이다.Therefore, in another aspect, the present invention provides a gene construct comprising a gene encoding the target protein and an EDA gene represented by SEQ ID NO: 34, or an EDA gene represented by SEQ ID NO: 34 As a fusion partner, a recombinant microorganism into which an expression vector for producing a target protein is introduced to induce the expression of the recombinant protein, and recovering the recombinant protein.
본 발명에 있어서, 상기 방법은 상기 재조합 단백질로부터 EDA를 제거하는 단계를 추가로 포함하는 것을 특징으로 할 수 있다.In the present invention, the method may further include the step of removing EDA from the recombinant protein.
본 발명의 EDA를 융합파트너로 사용하여 재조합 단백질을 생산하는 방법은 기존의 융합파트너가 가지고 있는 수용성과 접힘에 관한 한계를 극복할 수 있고, 단백질 의약품 및 산업용 생산에 폭넓게 이용될 수 있는 특징이 있다.
The method of producing the recombinant protein using the EDA of the present invention as a fusion partner can overcome limitations on the water solubility and folding of existing fusion partners and can be widely used for protein medicine and industrial production .
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1: 환경 스트레스에 의한 대장균 단백질체 변화 분석Example 1: Analysis of E. coli protein body change by environmental stress
융합파트너가 가져야 할 조건으로는 높은 발현률, 효과적인 3차원 구조형성 등을 들 수 있다. 특히 효과적인 3차원 구조를 형성하기 위해서는 단백질의 구조적 안정성이 전제되어야 한다. 이에, 생산 균주의 발현 환경에 변화를 유도하고 발현량 및 그 기능성이 유지되는 단백질을 분석하였다.
The conditions that fusion partners should have are high expression rates and effective three-dimensional structure formation. Particularly, in order to form an effective three-dimensional structure, the structural stability of the protein must be assumed. Thus, the expression level of the production strain and the protein whose function was maintained were analyzed.
1-1 : 단백질체 분석을 위한 수용성 단백질 준비1-1: Preparation of water-soluble proteins for protein analysis
단백질체 분석을 위해 대장균 BL21(DE3) 균주를 선택하고, 대장균에 단백질의 올바른 접힘을 저해하는 스트레스를 주기 위해 48℃에서 배양한 대장균에서 발현한 수용성 단백질을 회수하였다.Escherichia coli BL21 (DE3) strain was selected for proteomic analysis and a soluble protein expressed in Escherichia coli cultured at 48 ° C was recovered to give stress to inhibit proper folding of the protein in E. coli.
1L당 트립톤 10 g, 효모 추출물 10 g, NaCl 5 g으로 조성된 LB 배지에서 37℃, 130 rpm으로 배양한 대장균 BL21(Escherichia coli K-12)을 OD600이 0.5에 도달했을 때 37℃(대조군) 및 48℃(실험군)에서 3시간 더 배양한 후 세포배양액을 6000 rpm 4℃ 에서 원심 분리하여 균체 침전물을 회수하였다. pH 8.0, 40 mM 트리스 버퍼 (Tris buffer)로 2회 세척한 후, 세척된 대장균 침전물을 500 ㎕의 파쇄 용액 (lysis buffer; 8 M 우레아(urea), 4% (w/v) 챕스(CHAPS), 40 mM 트리스(Tris), 단백질 분해효소 제한 혼합물(protease inhibitor cocktail))에 현탁한 후 초음파 파쇄기(Branson sonifier, USA)를 이용하여 파쇄하였다. 균체 파쇄 후 12,000 rpm, 4℃ 에서 60분간 원심분리 하여 균체 파쇄물을 제거한 뒤 상등액을 분리하였다.
When E. coli BL21 (Escherichia coli K-12) was cultured in LB medium composed of 1L tryptone 10 g sugar, yeast extract 10 g, NaCl 5 g to 37 ℃, 130 rpm OD 600 reaches 0.5 37 ℃ ( (Control group) and 48 째 C (experimental group) for 3 hours, and the cell culture was centrifuged at 6000 rpm at 4 째 C to recover cell mass. After washing twice with 40 mM Tris buffer (pH 8.0), the washed E. coli precipitate was suspended in 500 μl of a lysis buffer (8 M urea, 4% w / v CHAPS, , 40 mM Tris, protease inhibitor cocktail) and disrupted using an ultrasonic disrupter (Branson sonifier, USA). After disruption of the cells, the cells were centrifuged at 12,000 rpm and 4 ° C for 60 minutes to remove cell lysate, and the supernatant was separated.
1-2 : 단백질체 분석을 위한 2차원 겔 전기영동 (2-dimensional gel electrophoresis)1-2: Two-dimensional gel electrophoresis for proteome analysis
분리된 상등액의 단백질 농도는 바이오-라드 단백질 분석 키트 (Bio-Rad protein assay kit)를 이용하여 측정하였다. 30 mg의 수용성 단백질을 포함하고 있는 상등액을 분획하여 재수화 용액(rehydration solution; 2 M 싸이오우레아(thiourea), 8 M 우레아(urea), 4 % w/v 챕스(CHAPS), 1 % w/v DTT, 1 % w/v 이동성 양성전해질(carrier ampholyte), pH 4.7)에 현탁 하였다. 1차원 등전점 분리과정은 바이오-라드 단백질 IEF cell 전기영동장치 (Bio-Rad Protein IEF cell electrophoresis system)를 이용하였으며, 선형 pH4-7 범위의 IPG(immobilized pH gradient) 겔 스트립(17 cm, ReadyStrip)을 이용하여 재수화 용액에 포함되어 있는 단백질을 하룻밤 동안 재수화 하였다. 500 V에서 2시간, 1000 V에서 30분, 2000 V에서 30분, 4000 V에서 30분, 8000 V에서 70000 VHr 동안 진행하였다. 재수화된 IPG 겔 스트립은 1 % DTT를 포함하고 있는 평형화 용액 (epuilibration solution; 50 mM 트리스(Tris), pH 8.6, 6 M 우레아(urea), 30 % v/v 글리세롤(glycerol), 2 % SDS, 약간의 브로모페놀 블루(bromophenol blue)에서 15분간 반응시킨 뒤 2.5 % 아이오도아세트아마이드(iodoacetamide)가 포함된 평형화 용액에서 15분간 반응시켰다. 평형화된 겔 스트립은 12.5% 폴리아크릴아마이드 겔을 이용하여 2차원 전기영동 한 뒤, 은-염색(silver staining)을 통해서 단백질 스팟을 검출한다. 염색된 겔은 GS-710 밀도계(densitometer)를 이용하여 스캐닝 한 후 PDQuest 소프트웨어 버전 6.1을 이용하여 단백질 스팟을 확인하였다. 단백질 스팟의 부피를 측정하여 비교 대장균에서 분리한 단백질을 기준으로 GdnHCl과 2-HEDS가 첨가된 대장균 단백질의 발현량을 상대적으로 비교 분석한 뒤, 스팟 부피가 증가한 단백질을 동정하였다.
The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit. The supernatant containing 30 mg of water soluble protein was fractionated and resuspended in a rehydration solution (2 M thiourea, 8 M urea, 4% w / v CHAPS, 1% w / v DTT, 1% w / v mobility carrier ampholyte, pH 4.7). One-dimensional isoelectric point separation was performed using a Bio-Rad Protein IEF cell electrophoresis system and an IPG (immobilized pH gradient) gel strip (17 cm, ReadyStrip) in a linear pH range of 4-7 The proteins contained in the rehydrated solution were rehydrated overnight. 2 hours at 500 V, 30 minutes at 1000 V, 30 minutes at 2000 V, 30 minutes at 4000 V, and 70000 VHr at 8000 V. The rehydrated IPG gel strips were washed with an epuilibration solution (50 mM Tris, pH 8.6, 6 M urea, 30% v / v glycerol, 2% SDS , Bromophenol blue for 15 minutes, and then reacted for 15 minutes in an equilibration solution containing 2.5% iodoacetamide. The equilibrated gel strips were applied using a 12.5% polyacrylamide gel The stained gel was scanned using a GS-710 densitometer and analyzed using a PDQuest software version 6.1 to determine the protein spot The volume of protein spot was measured and the expression level of GdnHCl and 2-HEDS-added Escherichia coli protein was compared and analyzed based on the protein isolated from the comparative E. coli. It was identified.
실시예 2 : MALDI-TOF-MS 분석 및 단백질 동정Example 2: MALDI-TOF-MS analysis and protein identification
MALDI-TOF-MS 분석을 위해서 실시예 1에서 선정된 비교 대장균의 단백질 보다 증가한 단백질을 은-염색된 겔로부터 추출하였다. 추출 된 단백질 스팟을 25 nM 암모늄 바이카보네이트(ammonium bicarbonate, pH 8.0) 용액에서 트립신(trypsin, 10.15 mg/ml) 분해 과정을 37℃ 에서 하룻밤 동안 진행하였다. 분해된 펩타이드는 5 % v/v TFA, 50 % v/v ACN 용액을 이용하여 추출하였으며, 이 과정을 세 번 반복한 뒤 진공 원심분리기를 이용하여 건조시켰다. 건조된 펩타이드를 50 % ACN / 0.1 % TFA 용액에 용해시킨 뒤 MALDI-TOF-MS 시스템 (MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems))을 이용하여 분석하였다. 분석된 펩타이드 질량 지문 (peptide mass fingerprints)은 Prospector 웹사이트의 MS-FIT (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm)을 이용하여 수행하였으며, 단백질 동정을 위한 MS-FIT 데이터 베이스는 Swiss-Prot을 이용하였다. For MALDI-TOF-MS analysis, proteins increased from the comparable E. coli protein selected in Example 1 were extracted from the silver-stained gel. The extracted protein spot was subjected to trypsin (10.15 mg / ml) digestion process in 25 nM ammonium bicarbonate (pH 8.0) solution overnight at 37 ° C. The digested peptides were extracted with 5% v / v TFA and 50% v / v ACN solution. This procedure was repeated three times and dried using a vacuum centrifuge. The dried peptides were dissolved in 50% ACN / 0.1% TFA solution and analyzed using a MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems). The analyzed peptide mass fingerprints were performed using the MS-FIT (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) on the Prospector website. MS-FIT The database was Swiss-Prot.
단백질 동정을 수행한 결과, 열충격 (heat shok) 스트레스 조건에서도 발현량이 1.65배 이상 증가하고 구조적 안정성을 유지하여, 스트레스 내성을 보이는 단백질은 EDA (E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase :E. coli KDPG aldolase)로 확인되었다 (표 1).As a result of the protein identification, the expression amount increased more than 1.65 times under the heat shok stress condition and the structural stability was maintained, and the protein showing stress tolerance was EDA ( E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase: E. coli KDPG aldolase) (Table 1).
유전자 이름
Gene name
단백질 이름
Protein name
서열 유사성(%)
Sequence similarity (%)
EDA
EDA
P0A955
P0A955
5.57/22.28
5.57 / 22.28
5.89/20.97
5.89 / 20.97
20%
20%
a. 유전자 접근 번호 : ExPASy Proteomics Server (http://www.expasy.org/)에서 유전자 정보를 검색하기 위한 식별번호a. Genetic access number: An identification number for retrieving genetic information from the ExPASy Proteomics Server ( http://www.expasy.org/ )
b. 이론값은 Compute pI/Mw tool (http://www.expasy.org/tools/pi_tool.html)을 이용하여 수집하였다. b. Theoretical values were collected using the Compute pI / Mw tool (http://www.expasy.org/tools/pi_tool.html).
c. 실험값은 이차원 전기영동 젤 이미지로부터 산출하였다.
c. Experimental values were calculated from two - dimensional electrophoresis gel images.
실시예 3 : 아미노 말단에 EDA를 융합파트너로 포함하는 발현 벡터의 제조Example 3: Preparation of expression vector containing EDA as a fusion partner at the amino terminus
실시예 1 내지 2의 방법으로 선정된 환경 스트레스 하에 수용성 발현량이 증가한 대장균 단백질 EDA(E. coli 2- keto-3-deoxy-6-phosphogluconate aldolase: E. coli KDPG aldolase)를 융합파트너로 포함하는 발현 벡터를 제조하였다.Expression of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase ( E. coli KDPG aldolase) with increased water-soluble expression level under environmental stresses selected by the methods of Examples 1 and 2 as fusion partners Vector.
EDA 유전자를 암호화하는 뉴클레오티드를 획득하기 위해, Entrez Nucleotide 데이타베이스의 gi:49175990에서 1877833bp 내지 1878474bp의 서열정보(서열번호 34)를 이용하여 정지 코돈을 제외한 EDA 유전자의 PCR 증폭을 위한 프라이머쌍을 제작하였다. 또한, 상기 프라이머쌍의 센스 프라이머(서열번호 1: cat atg aaa aac tgg aaa aca agt gca)에는 NdeI 제한효소 인식서열을, 안티센스 프라이머(서열번호 2: ctc gag cag ctt agc gcc ttc tac)에는 XhoI 제한효소 인식서열을 포함하도록 제작하였다. PCR은 DNA 중합효소반응용 완충용액(0.25 mM dNTPs; 50 mM KCl; 10 mM (NH4)2SO4; 20 mM Tris-HCl(pH8.8); 2 mM MgSO4; 0.1% Triton X-100)에 대장균으로부터 분리한 염색체를 주형 DNA로 100 ng, 서열번호 1 내지 2로 표시되는 프라이머쌍을 각각 50 pmol을 넣은 다음 Taq DNA 중합효소를 이용하여 수행하였다. 반응 조건은 95℃/30초(변성), 52℃/30초(어닐링), 72℃/60초(신장)로 총 30회 수행하였으며, 그 결과 증폭된 DNA절편의 5' 말단에 NdeI 제한효소 부위와 3' 말단에 XhoI 제한 효소 부위를 포함하는 PCR 산물을 수득하였다. 증폭된 PCR 산물을 제한효소 NdeI과 XhoI으로 각각 처리하여 pT7-7(Novagen, USA)의 제한효소 NdeI과 XhoI자리에 삽입하였고 이를 pT7-EDA 라고 명명하였다.
In order to obtain nucleotides encoding the EDA gene, primer pairs for PCR amplification of the EDA gene except the stop codon were prepared using 1877833 bp to 1878474 bp sequence information (SEQ ID NO: 34) at gi: 49175990 in the Entrez Nucleotide database . In addition, the sense primer of the primer pair (SEQ ID NO: 1: cat atg aaa aac tgg aaa aca agt gca), the NdeI restriction enzyme recognition sequence, the antisense primer (SEQ ID NO: 2: ctc gag cag ctt agc gcc ttc tac) is XhoI restriction Enzyme recognition sequence. The PCR was performed using a buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH 4 ) 2 SO 4 ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO 4 ; 0.1% Triton X-100 ), 100 ng of the template DNA and 50 pmol of each of the primer pairs shown in SEQ ID NOS: 1 to 2 were added to the chromosome from Escherichia coli and then Taq DNA polymerase was used. The reaction conditions were 30 times in total at 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing) and 72 ° C for 60 seconds (elongation). As a result, NdeI restriction enzyme And a XhoI restriction enzyme site at the 3 ' end. The amplified PCR products were treated with restriction enzymes NdeI and XhoI , respectively, and inserted into the restriction enzymes NdeI and XhoI of pT7-7 (Novagen, USA), which were named pT7-EDA.
실시예 4 : 목적 단백질의 단독발현 벡터 및 EDA 융합발현 벡터 제조Example 4: Production of a single expression vector and EDA fusion expression vector of a target protein
EDA를 융합발현파트너로 이용하여 다양한 목적 단백질의 수용성 발현 유도를 확인하기 위해 목적 단백질의 단독 발현, 융합 파트너와의 융합 발현을 위한 벡터 및 단백질 절단효소 인식부위를 포함하는 융합 발현 벡터를 제조하였다.
In order to confirm the induction of aqueous expression of various target proteins by using EDA as a fusion expression partner, a fusion expression vector containing a vector for expression of a target protein and a fusion expression partner with a fusion partner and a protein cleavage enzyme recognition site was prepared.
4-1 : 목적 단백질의 단독발현 벡터4-1: Single expression vector of the target protein
본 발명에 사용된 목적 단백질은 인간 프리프로-그렐린 (human prepro-ghrelin, 이하 'ppGRN'; 서열번호 35), 인간 인터루킨-2(human interleukin-2, 이하 'IL-2'; 서열번호 36), 인간 미니프로인슐린 (human minipro-insulin, 이하 'mp-INS'; 서열번호 37), 인간 상피세포 성장인자 (human epidermal growth factor, 이하 'EGF'; 서열번호 38), 인간 페리틴 가벼운 사슬(human ferritin light chain, 이하 'hFTN-L'; 서열번호 39), 인간 과립구 집락-자극인자(human granulocyte colony-stimulating factor, 이하 'G-CSF'; 서열번호40) 및 아르기닌 디이미나아제(Arginine deiminase, 이하 'ADI'; 서열번호 41) 이다. 목적 단백질의 주형 DNA는 상기 단백질들을 많이 발현하는 인간 조직으로부터 RNeasy mini kit(QIAGEN, USA)를 이용하여 전체 RNA를 추출한 뒤, 전체 RNA 1㎍과 oligo-d(T) 1㎕ (Invitrogen, USA, 0.5 ㎍/㎕)에 증류수를 50㎕ 까지 채운 후, AccuPower RTpremix(Bioneer, 한국)에 넣어 RT-PCR(reverse transcription polymerasechain reaction) 반응시켰다. 70℃에서 5분, 4℃에서 5분, 42℃에서 60분, 94℃에서 5분, 4℃에서 5분간 반응하여 cDNA를 합성함으로써 수득하였다. 상기 hIL-2, hFTN-L 및 G-CSF는 인간 백혈구, mp-INS는 인간 췌장 조직, EGF는 상피세포, ppGRN은 인간태반에서 각각 클로닝 하였다.The objective protein used in the present invention is human prepro-ghrelin (hereinafter abbreviated as' ppGRN '; SEQ ID NO: 35), human interleukin-2 (hereinafter referred to as' IL- , Human minipro-insulin (hereinafter abbreviated as 'mp-INS', SEQ ID NO: 37), human epidermal growth factor (EGF) (SEQ ID NO: 38), human ferritin light chain human granulocyte colony-stimulating factor (G-CSF) (SEQ ID NO: 40) and arginine deiminase (hereinafter abbreviated as " hGFN- Hereinafter " ADI "; SEQ ID NO: 41). The template DNA of the target protein was obtained by extracting total RNA from human tissues expressing the above proteins with a RNeasy mini kit (QIAGEN, USA), adding 1 μg of total RNA and 1 μl of oligo-d (T) (Invitrogen, USA, 0.5 μg / μl) was filled up to 50 μl of distilled water, and then subjected to reverse transcription polymerase chain reaction (RT-PCR) in an AccuPower RTpremix (Bioneer, Korea). Followed by 5 minutes at 70 ° C, 5 minutes at 4 ° C, 60 minutes at 42 ° C, 5 minutes at 94 ° C, and 5 minutes at 4 ° C to synthesize cDNA. The hIL-2, hFTN-L and G-CSF were cloned in human leukocytes, mp-INS in human pancreatic tissue, EGF in epithelial cells and ppGRN in human placenta respectively.
상기 목적 단백질의 단독 발현벡터를 제조하기 위해서 각 목적 단백질의 5'-말단에 NdeI, 3'-말단에 HindIII 제한효소 인지부위를 포함하도록 중합효소연쇄반응(polymerease chain reaction)하였다. 중합효소연쇄반응(polymerease chain reaction)의 조건은, [표 2]의 프라이머쌍을 각각 50 pmol씩 포함한 DNA 중합효소반응용 완충용액(0.25 mM dNTPs; 50 mM KCl; 10 mM (NH4)2SO4 20 mM Tris-HCl(pH8.8); 2 mM MgSO4 0.1% Triton X-100)에 주형 DNA 100 ng을 넣은 다음 Taq DNA 중합효소를 이용하여 95℃/30초(변성), 52℃/30초(어닐링), 72℃/60초(신장)로 총 30회 PCR을 수행하였다. 구체적으로, hIL-2, hFTN-L, G-CSF, AID, ADI 및 CUT는 시작 코돈을 제외하고 정지 코돈을 포함하는 아미노산 서열을 증폭하였고, GAD448-585의 경우에는 448번째 아미노산부터 585번째 아미노산 서열을 증폭하였다. 이때, GAD448-585의 경우는 3'-말단에 ClaI 제한효소 인식서열을 포함하도록 프라이머를 제작하였다.In order to prepare a single expression vector of the target protein, a polymerease chain reaction was performed so as to include NdeI at the 5'-end of each target protein and HindIII restriction enzyme recognition site at the 3'-end. The conditions of the polymerease chain reaction were as follows: 20 mM buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH 4) 2
상기 증폭된 목적 단백질을 코딩하는 NdeI/HindIII 단편을 잘라낸 후 대장균 발현벡터 pT7(Novagen) 벡터의 NdeI/HindIII 자리에 삽입하여 단백질 단독발현 벡터를 제작하였다(도 1).
The NdeI / HindIII fragment encoding the amplified target protein was cut out and inserted into the NdeI / HindIII site of the E. coli expression vector pT7 (Novagen) vector to prepare a protein-only expression vector (FIG. 1).
4-2 : 목적 단백질의 융합파트너와의 융합발현 벡터4-2: Fusion expression vector of target protein with fusion partner
ppGRN, IL-2, mp-INS, EGF, hFTN-L, hG-CSF 및 ADI 등의 목적 단백질의 5'-말단에 XhoI 제한효소 인식서열과 3'-말단에 HindIII 제한효소 인식서열을 포함하도록 제작된 [표 3]의 프라이머쌍을 각각 50 pmol씩 포함한 DNA 중합효소반응용 완충용액(0.25 mM dNTPs; 50 mM KCl; 10 mM (NH4)2SO4; 20 mM Tris-HCl(pH8.8); 2 mM MgSO4; 0.1% Triton X-100)에 주형 DNA 100 ng을 넣은 다음 Taq DNA 중합효소를 이용하여 95℃/30초(변성), 52℃/30초(어닐링), 72℃/60초(신장)로 총 30회 PCR을 수행하였다. 구체적으로, ppGRN, IL-2, mp-INS, EGF, hFTN-L, hG-CSF 및 ADI는 시작 코돈을 제외하고 정지 코돈을 포함하는 아미노산 서열을 증폭하였다. ppGRN, IL-2, mp- INS, EGF, hFTN-L, hG-CSF , and to include a HindIII restriction enzyme recognition sequences for the restriction enzyme XhoI recognition sequence and the 3'-end to the 5'-terminal of the desired protein, such as ADI produced [Table 3] DNA polymerase reaction buffer, including for each of 50 pmol each of the primer pairs of the solution (0.25 mM dNTPs; 50 mM KCl ; 10 mM (NH 4) 2
상기 PCR 증폭 산물을 XhoI/HindIII 제한 효소로 처리한 후, 실시예 3의 방법으로 제작한 발현벡터 pT7-EDA의 XhoI/HindIII 자리에 삽입함으로써 외래단백질의 융합파트너와의 융합발현 벡터를 제작하였다(도 1). 상기의 방법으로 만들어진 플라스미드를 각각 EDA::ppGRN, EDA::IL-2, EDA::mp-INS, EDA::EGF, EDA::hFTN-L, EDA::hG-CSF 및 EDA::ADI 라고 명명하였다.The PCR amplification product was treated with XhoI / HindIII restriction enzyme and inserted into the XhoI / HindIII site of the expression vector pT7-EDA prepared by the method of Example 3 to prepare a fusion expression vector with a fusion partner of the foreign protein ( 1). The plasmids prepared by the above method are referred to as EDA :: ppGRN, EDA :: IL-2, EDA :: mp-INS, EDA :: EGF, EDA :: hFTN-L, EDA :: hG- .
또한, 기존에 효과적인 융합파트너라고 알려져 있는 글루타치온-S-전이효소(Glutathione-S-transferase, 이하 'GST')을 각각 7종류의 목적단백질의 아미노 말단에 융합파트너로 삽입한 뒤 GST::ppGRN, GST::IL-2, GST::mp-INS, GST::EGF, GST::hFTN-L, GST::hG-CSF 및 GST::ADI를 제조하였다.
In addition, glutathione-S-transferase (GST), previously known as an effective fusion partner, was inserted as a fusion partner in the amino terminal of seven target proteins, respectively, and GST :: ppGRN, GST :: hG-CSF, and GST :: ADI were prepared from GST :: IL-2, GST :: mp-INS, GST :: EGF, GST :: hFTN-L.
4-3 : 6개의 히스티딘 및 단백질 절단효소 부위를 포함하는 정제용 발현 벡터4-3: Expression vector for purification comprising six histidine and protein cleavage enzyme sites
EDA가 융합발현된 hG-CSF, hFTN-L을 정제하기 위해서, EDA의 5'-말단에 6개의 히스티딘을 포함하며 EDA 융합파트너와 목적단백질 사이에 엔테로카이네이즈(enterokinase)에 의해 인지되어 절단될 수 있는 시퀀스 D4K(DDDDK)를 포함하는, 다음과 같은 정제용 발현벡터를 제작하였다. In order to purify hG-CSF, hFTN-L, in which EDA is fused, EDTA contains six histines at the 5'-end and is cleaved and recognized by enterokinase between the EDA fusion partner and the target protein The following expression vector for purification, containing the sequence D 4 K (DDDDK), was prepared.
EDA의 5'-말단에 6개의 히스티딘(histidine, 이하 'His6')과 NdeI, 3'-말단에 XhoI 제한효소 인지부위를 포함하도록, 센스 프라이머(서열번호 31: cat atg cac cat cac cat cac cat aaa aac tgg aaa aca agt gca) 및 안티센스 프라이머(서열번호 2: ctc gag cag ctt agc gcc ttc tac)를 50 pmol씩 포함한 DNA 중합효소반응용 완충용액(0.25 mM dNTPs; 50 mM KCl; 10 mM (NH4)2SO4 20 mM Tris-HCl(pH8.8); 2 mM MgSO4 0.1% Triton X-100)에 주형 DNA 100 ng을 넣은 다음 Taq DNA 중합효소를 이용하여 95℃/30초(변성), 52℃/30초(어닐링), 72℃/60초(신장)로 총 30회 중합효소연쇄반응(polymerease chain reaction)을 수행하였다. 증폭된 정제용 His6-EDA을 코딩하는 NdeI/XhoI 단편을 잘라낸 후 목적 단백질(ADI, hG-CSF 및 hFTN-L)이 포함되어 있는 발현 벡터의 NdeI/XhoI 자리에 삽입하였고, 이를 H6::EDA::ADI, H6::EDA::hG-CSF 및 H6::EDA::hFTN-L로 명명하였다.A sense primer (SEQ ID NO: 31) was prepared so as to include six histidine (His6 ') and NdeI at the 5'-end of EDA and a XhoI restriction enzyme recognition site at the 3'- (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH)) containing 50 pmol of an antisense primer (aaa aac tgg aaa aca agt gca) and an antisense primer (SEQ ID NO: 2: ctc gag cag ctt agc gcc ttc tac) 4) 2 SO 4 20 mM Tris -HCl (pH8.8); 2
또한, 융합단백질(EDA)로부터 목적단백질(hFTN-L, hG-CSF)을 분리해 내기 위하여 목적단백질의 5'-말단에 엔테로카이네이즈(enterokinase)에 의해 인지되어 절단될 수 있는 시퀀스와 XhoI 제한효소 인지부위, 3'-말단에 HindIII 제한효소 인지부위를 포함하도록, hFTN-L 융합 폴리펩티드를 코딩하는 폴리뉴클레오티드 단편의 센스 프라이머(서열번호 32: ctc gag gat gac gat gac aag agc tcc cag att cgt), 안티센스 프라이머(서열번호 26: aag ctt tta gtc gtg ctt gag agt) 및 hG-CSF 융합 폴리펩티드를 코딩하는 폴리뉴클레오티드 단편의 센스 프라이머(서열번호 33: ctc gag gac gat gac gat aaa acc ccc ctg ggc cct gcc), 안티센스 프라이머(서열번호 28: aag ctt tca tgg ctg tgc aag)를 50 pmol씩 포함한 DNA 중합효소반응용 완충용액(0.25 mM dNTPs; 50 mM KCl; 10 mM (NH4)2SO4 20 mM Tris-HCl(pH8.8); 2 mM MgSO4 0.1% Triton X-100)에 주형 DNA 100 ng을 넣은 다음 Taq DNA 중합효소를 이용하여 95℃/30초(변성), 52℃/30초(어닐링), 72℃/60초(신장)로 총 30회 중합효소연쇄반응(polymerease chain reaction)을 수행하였다. 증폭된 엔테로카이네이즈(enterokinase) 인지 씨퀀스-목적단백질을 코딩하는 XhoI/HindIII 단편을 잘라낸 후 H6::EDA 단백질이 포함되어 있는 발현 벡터의 XhoI/HindIII 자리에 삽입하였고, 이를 H6::EDA::EK::hG-CSF 및 H6::EDA::EK::hFTN-L로 명명하였다(도 1).
It is also an object from the fusion protein (EDA) protein (hFTN-L, hG-CSF ) is aware of the enterovirus by a kinase (enterokinase) to the 5'-terminal of the desired protein in order to separate the sequence and XhoI restriction enzymes, which may be cut A sense primer (SEQ ID NO: 32: ctc gag gat gac gat gac aag agc tcc cag att cgt) of the polynucleotide fragment encoding the hFTN-L fusion polypeptide, including a HindIII restriction enzyme recognition site at the 3'- (SEQ ID NO: 33: ctc gag gac gat gac gat aaa acc ccc ctg ggc cct gcc) of an antisense primer (SEQ ID NO: 26: aag ctt tta gtc gtg ctt gag agt) and a polynucleotide fragment encoding hG- 50 mM KCl; 10 mM (NH 4 ) 2 SO 4 20 mM Tris-HCl buffer solution containing 50 pmol of an antisense primer (SEQ ID NO: 28: aag ctt tgg ctg tgc aag) HCl (pH 8.8); 2 mM MgSO 4 0.1% Triton X-100) (denaturation) at 95 ° C for 30 sec (denaturation), 52 ° C for 30 sec (annealing) and 72 ° C / 60 sec (elongation) using Taq DNA polymerase for 30 cycles of polymerease chain reaction Respectively. The XhoI / HindIII fragment encoding the amplified enterokinase sequence-target protein was cut out and inserted into the XhoI / HindIII site of the expression vector containing the H6 :: EDA protein. This was inserted into the H6 :: EDA :: EK :: hG-CSF and H6 :: EDA :: EK :: hFTN-L (Fig. 1).
실시예 5 : 재조합 단백질의 수용성 발현Example 5: Aqueous expression of recombinant protein
실시예 4의 방법으로 제조한 목적단백질의 단독발현 벡터 및 융합파트너와의 융합발현 벡터를 대장균에 형질전환하여 배양한 뒤, IPTG로 재조합 단백질의 발현을 유도함으로써 본 발명의 융합파트너에 의한 수용성 발현의 효과를 확인하였다.The expression of the recombinant protein by the IPTG was induced by transforming Escherichia coli into a single expression vector and a fusion partner expression vector of the objective protein prepared in Example 4, .
하나한(Hanahan)이 기술한 방법(Hanahan D, DNA Cloning vol.1 109-135, IRS press, 1985)에 의해 실시예 4의 벡터들을 대장균에 형질전환 하였다. 구체적으로 CaCl2로 처리한 대장균 BL21(DE3)에 실시예 4의 벡터들을 열충격 방법으로 형질전환시킨 후, 앰피실린(ampicillin)이 포함된 배지에서 배양하여 상기 발현벡터가 형질전환되어 앰피실린 저항성을 나타내는 콜로니를 선별하였다. 융합파트너와의 융합발현 벡터 pT7-EDA로 형질전환된 대장균을 BL21(DE3):pT7-EDA로 명명하였다. 상기 콜로니를 하룻밤 동안 LB 배지에서 배양한 종균 배양액의 일부를 100 mg/ml 앰피실린을 포함하는 LB 배지에 접종한 다음 37℃에서 130 rpm으로 배양하였다. 배양액의 OD600이 0.5에 이르렀을 때 IPTG를 첨가(1 mM)하여 재조합 유전자의 발현을 유도하였다. IPTG 첨가 후 동일한 조건(37℃)으로 4시간 더 배양하거나, 20℃에서 130rpm으로 12시간 동안 배양하였다.The vectors of Example 4 were transformed into E. coli by the method described by Hanahan (Hanahan D, DNA Cloning vol. 109-135, IRS press, 1985). Specifically, the vectors of Example 4 were transformed into Escherichia coli BL21 (DE3) treated with CaCl 2 by a thermal shock method and then cultured in a medium containing ampicillin to transform the expression vector into ampicillin resistance Were selected. The fusion E. coli transformed with the expression vector pT7-EDA was named BL21 (DE3): pT7-EDA. A portion of the culture medium in which the colonies were cultured in LB medium overnight was inoculated into LB medium containing 100 mg / ml ampicillin and cultured at 37 ° C at 130 rpm. When the OD 600 of the culture reached 0.5, IPTG was added (1 mM) to induce the expression of the recombinant gene. After addition of IPTG, the cells were further cultured under the same conditions (37 ° C) for 4 hours or cultured at 20 ° C and 130 rpm for 12 hours.
상기 방법으로 배양한 대장균을 13,000 rpm으로 5분간 원심분리하여 균체 침전물을 회수한 후 5 ml의 파쇄 용액(10 mM Tris-HCl 완충액, pH 7.5, 10 mM EDTA)에 현탁하여 초음파 파쇄기(Branson sonifier, Branson Ultrasonics Corporation, USA)를 이용하여 파쇄하였다. 파쇄한 후 13,000 rpm으로 10분간 원심분리한 뒤 상등액과 균체 파쇄물을 분리하였다. 균체 파쇄물은 1% 트리톤 엑스 100 (Triton X-100)을 이용하여 2회 세척하였다. 분리된 상등액의 단백질 농도는 바이오-라드 단백질 분석 키트(Bio-Rad protein assay kit, USA)를 이용하여 측정하였다. 상등액과 균체 파쇄물을 각각 5 X SDS(0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% 글리세롤, 37.5 mM DTT)과 1:4로 섞어 10% SDS-PAGE 겔의 웰에 로딩하고 125 V에서 2시간 동안 시료를 전개한 다음 겔을 쿠마시 염색 방법으로 염색한 후 탈색하여 각각의 재조합 단백질의 발현량을 밀도계(Densitometer, Duoscan T1200, Bio-Rad, USA)로 확인하고, 수학식 1에 따라 용해도(%)를 계산하였다.Escherichia coli cultured by the above method was centrifuged at 13,000 rpm for 5 minutes to collect the cell pellet, suspended in 5 ml of a disruption solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA) and sonicated using a Branson sonifier Branson Ultrasonics Corporation, USA). After crushing, the supernatant was separated from the cell lysate by centrifugation at 13,000 rpm for 10 minutes. The cell lysate was washed twice with 1% Triton X-100. The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit (USA). The supernatant and cell lysate were loaded into wells of 10% SDS-PAGE gel mixed with 1: 4 with 5 x SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) , The gel was stained with Coomassie staining method and decolorized. The expression amounts of the respective recombinant proteins were confirmed with a density meter (Densitometer, Duox T1200, Bio-Rad, USA) The solubility (%) was calculated according to the following formula.
그 결과, EDA와 융합한 목적단백질의 발현이 대부분 불용성 응집체보다 수용성 발현에서 더 많은 것을 확인하였으며, 또한 단독 발현한 외래단백질의 수용성 발현량보다 EDA와 융합한 외래단백질에서 용해도가 매우 증가하는 것을 확인하였다(도 2). 도 2에서 확인할 수 있듯이 대부분의 목적 단백질의 수용성 발현량이 증가하였으며, 이러한 결과는 단백질체 분석을 통해서 확보한 EDA가 융합파트너로써의 효과가 크다는 것을 의미한다.As a result, it was confirmed that the expression of the target protein fused with EDA was more than that of the insoluble aggregate more than that of the insoluble aggregate, and that the solubility was significantly increased in the exogenous protein fused with EDA than the soluble expression amount of the exogenous protein expressed alone (Fig. 2). As shown in FIG. 2, the water-soluble expression level of most target proteins was increased. These results indicate that the EDA obtained through the protein-protein analysis has a great effect as a fusion partner.
또한, 기존에 효과적인 융합파트너라고 알려져 있는 글루타치온-S-전이효소(Glutathione-S-transferase, 이하 'GST')을 각각 7종류의 목적단백질의 아미노 말단에 융합파트너로 삽입한 뒤 (GST::ppGRN, GST::IL-2, GST::mp-INS, GST::EGF, GST::hFTN-L, GST::hG-CSF 및 GST::ADI), 대장균 BL21(DE3) 균주에 형질전환하여 동일한 방법으로 발현 양상을 확인한 결과, EDA를 아미노 말단에 융합파트너로 사용한 것이 GST를 융합파트너로 사용한 것 보다 수용성 발현량의 증가 정도가 훨씬 더 뛰어남을 확인할 수 있었다 (도 2).
In addition, glutathione-S-transferase (GST), previously known as an effective fusion partner, was inserted into the amino terminus of seven kinds of target proteins as fusion partners (GST :: ppGRN , GST :: IL-2, GST :: mp-INS, GST :: EGF, GST :: hFTN-L, GST :: hG-CSF and GST :: ADI) and Escherichia coli BL21 (DE3) As a result of confirming the expression pattern by the same method, it was confirmed that the use of EDA as a fusion partner at the amino terminus was much better than that of using GST as a fusion partner (Fig. 2).
실시예 6 : 재조합 단백질의 고유의 구조 및 활성도 유지Example 6: Retention of inherent structure and activity of recombinant protein
6-1 : EDA 융합발현된 아르기닌 탈이민효소(ADI)의 활성도 측정6-1: Measurement of activity of EDA-fused arginine deaminase (ADI)
융합발현파트너는 목적 단백질을 수용성의 형태로 발현시키는 것도 중요하지만 고유의 3차 구조를 갖도록 유도하여 기능성을 갖춘 활성형 형태로 발현시키는 것이 무엇보다 중요하다. 이를 확인하기 위해 융합파트너 EDA와 목적단백질 ADI를 융합발현한 뒤 활성도를 측정하였다. It is important that the fusion expression partner is expressed in a water-soluble form, but it is important to induce the fusion protein to have an inherent tertiary structure and to express it in an active form having a functional property. To confirm this, the fusion partner EDA and the target protein ADI were fused and expressed, and the activity was measured.
미생물 유래의 아르기닌 탈이민효소(ADI)는 L-아르기닌을 L-시트룰린으로 전환시킨다. 이러한 특성을 이용하여 실시예 4를 통해서 확보한 EDA가 융합발현된 활성형 ADI의 활성도를 확인하기 위해서 다음과 같은 분석을 수행하였다. The microorganism-derived arginine deaminase (ADI) converts L-arginine to L-citrulline. Using these characteristics, the following assay was performed to confirm the activity of the fusion-expressed active ADI obtained by Example 4.
상기 EDA::ADI 발현 벡터를 대장균 BL21(DE3)에 형질전환한 후 37℃ 온도, LB 엠피실린 배양액에서 배양한 뒤 초음파 파쇄 방법으로 목적 단백질을 함유하는 추출물을 얻었다. 이렇게 얻은 EDA::ADI 융합발현 단백질에 대해서 100ml의 포스페이트 버퍼 (phosphate buffer, 0.1 M, pH 6.5.0)와 0.1742g L-아르기닌(L-arginine)을 혼합하여 10mM L-아르기닌 혼합액 만든 후, 혼합액 270㎕에 30㎕ EDA::ADI 상등액과 비교 실험으로 PBS 버퍼를 각각 첨가하였다. 반응은 첨가한 후 바로 시작되며, 반응이 시작되고 3분 간격으로 12분까지 530nm 파장에서 흡광도의 변화를 측정하였다. (Duoscan T1200, Bio-Rad, Hercules, CA).The EDA :: ADI expression vector was transformed into Escherichia coli BL21 (DE3) and cultured at 37 ° C in an LB-epicillin culture, followed by sonication to obtain an extract containing the desired protein. The EDA :: ADI fusion expression protein thus obtained was mixed with 100 ml of a phosphate buffer (0.1 M, pH 6.5.0) and 0.1742 g of L-arginine to prepare a 10 mM L-arginine mixed solution, PBS buffer was added to each of 270 쨉 l of 30 쨉 l EDA :: ADI supernatant as a comparative experiment. The reaction started immediately after addition and the change of absorbance at 530 nm wavelength was measured at the beginning of the reaction and every 3 minutes for 12 minutes. (Duoscan T1200, Bio-Rad, Hercules, Calif.).
그 결과, EDA::ADI 융합발현 단백질은 L-아르기닌이 L-시트룰린으로 변화되어 활성 형태로 발현됨을 확인하였다 (도 4).
As a result, it was confirmed that the EDA :: ADI fusion expression protein was changed to L-citrulline and expressed as an active form (FIG. 4).
6-2 : EDA 융합발현을 통해 생산된 hG-CSF와 hFTN-L의 정제 및 분석6-2: Purification and analysis of hG-CSF and hFTN-L produced by EDA fusion expression
hG-CSF는 의학용으로 주로 사용됨에 따라 항원항체 반응을 피하기 위해 융합발현파트너는 hG-CSF로부터 분리되어야 한다. 또한, hG-CSF와 같은 단백질은 인체 내에서는 먼저 전구체로 만들어지며 이 전구체가 프로티아제에 의한 성숙과정을 거쳐 Thr-Pro-Leu의 N-말단 아미노산 서열을 가지는 hG-CSF로 만들어지지만, 대장균에서 발현시킬 경우 아미노 펩티디아제 효소역가에 따라 시작코돈(start codon)에 해당하는 메티오닌(methionine)이 N-말단에 존재하게 되어 경우에 따라 N-말단의 메티오닌이 제거되지 않은 단백질이 혼재하게 되는데 정제과정 중에 이의 제거가 매우 어렵고, 비활성의 불용성 단백질로 발현될 경우 활성을 가지는 수용성 형태로 만들어주는 과정을 거쳐야 하는데, 이때 수율이 떨어지는 단점이 있다. As hG-CSF is mainly used for medical purposes, the fusion expression partner must be isolated from hG-CSF to avoid antigen-antibody reaction. In addition, a protein such as hG-CSF is first made into a precursor in the human body, and this precursor is made into hG-CSF having an N-terminal amino acid sequence of Thr-Pro-Leu through a protease-induced maturation process, The methionine corresponding to the start codon is present at the N-terminus according to the amino-peptidase enzyme activity, resulting in a mixture of proteins in which N-terminal methionine has not been removed in some cases It is very difficult to remove it during the purification process, and when it is expressed as an insoluble insoluble protein, it must be made into a water-soluble form having an activity. At this time, however, the yield is inferior.
따라서, 본 발명에서는 엔테로카이네이즈(enterokinase)로 절단 되었을 때 천연형의 hG-CSF와 동일한 DNA를 가질 수 있게 만들어 줌으로서 이러한 단점을 극복할 수 있게 하였다. 상기 실시예 4를 통하여 구축한 발현 벡터 H6::EDA::EK::hG-CSF 및 H6::EDA::EK::hFTN-L으로 형질전환 된 대장균 BL21(DE3)를 이용하여 상기 실시예 5의 초음파 파쇄 단계까지 동일하게 시행하였다. EDA의 N-말단 쪽에는 6개의 histidine이 위치하게 되고 histidin과 Ni+2 금속의 친화력을 이용한 크로마토그래피를 통해 정제가 가능하게 하였다. Ni-NTA Agarose bead(QIAGEN)를 사용하여 Ni+2 금속 친화 크로마토그래피를 수행하였으며, 이를 통해, EDA::hG-CSF와 EDA::hFTN-L 을 각각 성공적으로 정제할 수 있었다.Therefore, in the present invention, it is possible to overcome this disadvantage by making it possible to have the same DNA as natural hG-CSF when it is cleaved by enterokinase. Using E. coli BL21 (DE3) transformed with the expression vectors H6 :: EDA :: EK :: hG-CSF and H6 :: EDA :: EK :: hFTN-L constructed in Example 4, 5 was performed until the ultrasonic disintegration step. Six histidine residues were located on the N - terminal side of EDA and purification was possible by chromatography using the affinity of histidine and Ni + 2 metal. Ni + 2 metal affinity chromatography was performed using Ni-NTA agarose bead (QIAGEN) to successfully purify EDA :: hG-CSF and EDA :: hFTN-L, respectively.
정제과정은 세포를 파쇄한 후 13,000rpm에서 10분간 원심분리한 다음 분리된 상등액을 금속친화를 이용한 정제를 위해서 Ni-NTA Agarose bead (QIAGEN) 500mL과 결합시켰다. Ni-NTA Agarose bead와 반응시키기 전에 결합 완충액 [binding buffer, pH 8.0, 50 mM 소듐 포스페이트(sodium phosphate), 300 mM 염화 나트륩 (NaCl), 10 mM 이미다졸(imidazole)]을 사용하여 세척하였다. His6-EDA-Enterokinase site-목적단백질(hG-CSF, hFTN-L)가 포함되어 있는 상등액과 Ni-NTA Agarose bead의 결합은 4℃에서 진행하고 2시간 이상 충분히 결합시킨 후 8 mL의 세척 완충액 [washing buffer, pH 8.0, 50 mM 소듐 포스페이트(sodium phosphate), 300 mM 염화 나트륨 (NaCl), 50 mM 이미다졸(imidazole)]을 이용하여 두 번 세척하였다. 다음 단계로 PBS 완충용액 [PBS buffer, 137mM 염화 나트륩(NaCl), 2.7 mM 염화 칼륨(KCl), 10 mM 제1인산나트륨(Na2HPO4), 2 mM 제1인산칼륨(KH2PO4), pH 7.4]을 이용하여 한번 더 추가 세척하였다. Agarose bead에 붙어 있는 His6-EDA-Enterokinase site-목적단백질(hG-CSF, hFTN-L)에 엔테로카이네이즈 용액 [enterokinase 5㎕, 10 X enterokinase buffer 50㎕, PBS 445㎕ (Invitrogen)] 500㎕을 첨가하여 22℃에서 12시간 반응시키고 elution fraction을 받아 13,000rpm에서 15분 동안 원심분리하여 수용성과 불용성 용액을 분리하였다. 분리한 샘플은 SDS-PAGE 분석을 통해 엔테로카이네이즈를 가하여 발현된 융합단백질(EDA::hG-CSF)로부터 EDA을 제거한 후에도 hG-CSF 또는 hFTN-L이 여전히 수용성 상태로 남아 있음을 확인하였고, 이렇게 정제된 hG-CSF는 mouse anti-human G-CSF monoclonal antibody (Clone 3D1, Santa Cruz Biotechnology Inc, CA)를 사용하여 Western 분석을 통해 hG-CSF가 확실함을 확인하였다(도 5).
After the cells were disrupted, the cells were centrifuged at 13,000 rpm for 10 minutes, and the separated supernatant was combined with 500 mL of Ni-NTA agarose bead (QIAGEN) for purification using metal affinity. Prior to reaction with Ni-NTA agarose beads, they were washed with binding buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole). Binding of the supernatant containing the His-EDTA-Enterokinase site-target protein (hG-CSF, hFTN-L) to the Ni-NTA Agarose bead proceeded at 4 ° C, washing buffer, pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole]. In the next step, PBS buffer, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM potassium phosphate monobasic (KH 2 PO 4 ), pH 7.4]. To the His6-EDA-Enterokinase site-target protein (hG-CSF, hFTN-L) attached to the agarose bead, 500 μl of an enterokinase solution (5 μl of enterokinase, 50 μl of 10 X enterokinase buffer, 445 μl of PBS (Invitrogen) The reaction mixture was reacted at 22 ° C for 12 hours, and the elution fraction was taken and centrifuged at 13,000 rpm for 15 minutes to separate the water-soluble and insoluble solution. It was confirmed that hG-CSF or hFTN-L still remains in a water-soluble state even after removing the EDA from the expressed fusion protein (EDA :: hG-CSF) by adding the enterokinase through SDS-PAGE analysis to the separated sample Western blot analysis of purified hG-CSF using mouse anti-human G-CSF monoclonal antibody (Clone 3D1, Santa Cruz Biotechnology Inc, CA) confirmed that hG-CSF was positive (FIG.
6-3 : 원평광 이색성 분광(CD) 분석을 통한 단백질 2차 구조 확인6-3: Identification of protein secondary structure by analysis of circular dichroism spectroscopy (CD)
융합발현파트너는 hG-CSF를 수용성의 형태로 발현시키는 것도 중요하지만 고유의 구조를 유지하도록 유도하여 발현시키는 것이 무엇보다 중요하기 때문에 이를 확인하기 위해 원평광 이색성 분광(CD) 분석으로 정제된 재조합 hG-CSF와 천연의 hG-CSF의 단백질 2차 구조의 변형여부를 비교/분석하였다. It is important to express hG-CSF in a water-soluble form, but it is important to induce and express the hG-CSF to maintain its inherent structure. Therefore, in order to confirm this fact, recombinant hG-CSF purified by a circularly dichroism spectroscopy (CD) CSF and natural hG-CSF were compared and analyzed.
실시예 6-2에서 정제된 재조합 hG-CSF에서 엔테로카이네이즈를 제거하기 위해, EK-AwayTMresin(Invitrogen, Germany, Cat. No. R180-01)를 사용하였으며, 엔테로카이네이즈가 99%이상 제거된 용액은 microcon YM-10 (Millipore, Ireland)으로 CD 분석을 위해 필요한 양 만큼 농축(0.4167㎍/L)하여 분석을 위한 샘플을 준비하였다. 준비된 재조합 hG-CSF 샘플은 한국기초과학지원연구원(오창)에 의뢰하여 JASCO J-710 spectropolarimeter에 의해 원평광 이색성 분광(CD) 분석하였다. 상기 방법으로 정제된 재조합 hG-CSF와 천연의 hG-CSF[Grasin Prefilled- Syringe (Filgrastim), Kirin, Japan)]의 단백질 2차구조를 비교분석 결과 정제된 재조합 hG-CSF는 천연형 hG-CSF와 같은 단백질 2차구조를 가짐을 확인하였다 (도 5).
EK-Away TM resin (Invitrogen, Germany, Cat. No. R180-01) was used to remove the enterokinase from the recombinant hG-CSF purified in Example 6-2, and 99% or more of the enterokinase The solution was concentrated (0.4167 ug / L) by the amount required for CD analysis with a microcon YM-10 (Millipore, Ireland) to prepare samples for analysis. The prepared recombinant hG-CSF samples were analyzed by a JASCO J-710 spectropolarimeter with a circularly polarized dichroism spectrometer (CD) with the Korea Basic Science Institute (Ochang). CSF and natural hG-CSF [Grasin Prefilled-Syringe (Filgrastim), Kirin, Japan)] as a result of a comparative analysis of the purified secondary recombinant hG- (Fig. 5).
6-4 : 투과전자 현미경 이미지 분석에 의한 hFTN-L 나노입자의 생성 확인6-4: Confirmation of production of hFTN-L nanoparticles by transmission electron microscopy image analysis
인간 유래의 훼리틴 단백질(hFTN-L)은 철 저장 단백질로 인간 훼리틴은 중쇄(heavy chain, 21kDa) 및 경쇄(light chain, 19kDa)로 구성되어 있으며, 24개의 단량체가 자가조립 과정을 통해서 12 nm의 균일한 직경을 갖는 구형 입자를 형성한다고 알려져 있다. 본 실시예에서는 융합발현파트너를 제거한 이후에도 구형의 입자를 형성하는지와 수용성 정도를 확인하였다.Human ferritin is composed of a heavy chain (21 kDa) and a light chain (19 kDa), and 24 monomers are self-assembled to form 12 lt; RTI ID = 0.0 > nm. < / RTI > In this Example, spherical particles were formed and the degree of water solubility was confirmed even after the fusion fusion expression partner was removed.
엔테로카이네이즈를 가하여 발현된 융합단백질(EDA::hFTN-L)로부터 EDA을 제거하고 난 뒤 hFTN-L의 수용성 정도를 SDS-PAGE 겔을 통하여 확인하였으며(도 6A), 또한 이렇게 정제된 hFTN-L을 투과전자현미경 (transmission electron microscopy, TEM) 분석을 하였다.After the EDA was removed from the expressed fusion protein (EDA :: hFTN-L) with the addition of enterokinase, the degree of water solubility of hFTN-L was confirmed by SDS-PAGE gel (Fig. 6A) Were analyzed by transmission electron microscopy (TEM).
투과전자현미경(transmission Electron Microscope, 이하 'TEM') 이미지 분석을 위해 상기 실시예 6-1에서 정제된 단백질 용액의 염색하지 않은 시료를 탄소 코팅 청동 전자 현미경 그리드(carbon-coated copper electron microscopy grid)에 공기 건조시킨 후 부착하였다. 단백질 나노입자의 염색된 이미지를 얻기 위해 2% 수성 우라닐 아세테이트(aqueous uranyl acetate) 용액(Ted Pella, 미국)에서 실온에서 10분간 방치하고, 증류수로 3 내지 4회 세척한 후, 단백질 나노입자 이미지는 Philips Technai 120kV 전자 현미경을 사용하여 관찰 하였다. For the transmission electron microscope (TEM) image analysis, an unstained sample of the protein solution purified in Example 6-1 was applied to a carbon-coated copper electron microscopy grid Air dried and then attached. To obtain a stained image of protein nanoparticles, they were left in a 2% aqueous uranyl acetate solution (Ted Pella, USA) at room temperature for 10 minutes, washed 3 to 4 times with distilled water, Were observed using a Philips Technai 120 kV electron microscope.
그 결과, 융합발현파트너(EDA)가 제거 된 이후에도, hFTN-L은 직경 12 nm 가량의 구형의 나노입자를 형성함을 확인할 수 있었다 (도 6B).
As a result, it was confirmed that even after the fusion fusion partner (EDA) was removed, hFTN-L formed spherical nanoparticles having a diameter of about 12 nm (FIG. 6B).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> Korea University Research and Business Foundation <120> Method for Preparing Recombinant Protein Using EDA as a Fusion Expression Partner <130> P15-B010 <160> 41 <170> KopatentIn 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> EDA-S <400> 1 catatgaaaa actggaaaac aagtgca 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EDA-AS <400> 2 ctcgagcagc ttagcgcctt ctac 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-S-NdeI <400> 3 catatgggct ccagcttcct g 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-AS-HindIII <400> 4 aagctttcac ttgtcggct 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-S-NdeI <400> 5 catatggcac ctacttcaag t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-AS-HindIIII <400> 6 aagcttttat caagtcagtg t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-S-NdeII <400> 7 catatgtttg tcaaccaaca t 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-AS-HinIII <400> 8 aagcttttag ttacagtagt tc 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-S-NdeI <400> 9 catatgaact ctgactccga atgc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-AS-HindIII <400> 10 aagcttttaa cgcagttccc acca 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-S-NdeI <400> 11 catatgagct cccagattcg t 21 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-AS-HindIII <400> 12 aagcttttag tcgtgcttga gagt 24 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-NdeI <400> 13 catatgactc cactcggacc tg 22 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-AS-HindIII <400> 14 aagctttcat ggctgtgcaa g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-NdeI <400> 15 catatgtctg tatttgacag t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-AS-HindIII <400> 16 aagcttctat cacttaacat c 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-S-XhoI <400> 17 ctcgagggct ccagcttcct g 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-AS <400> 18 aagctttcac ttgtcggct 19 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-S-XhoI <400> 19 ctcgaggcac ctacttcaag t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-AS <400> 20 aagcttttat caagtcagtg t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-S-XhoI <400> 21 ctcgagtttg tcaaccaaca t 21 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-AS <400> 22 aagcttttag ttacagtagt tc 22 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-S-XhoI <400> 23 ctcgagaact ctgactccga atgc 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-AS <400> 24 aagcttttaa cgcagttccc acca 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-S-XhoI <400> 25 catatgagct cccagattcg t 21 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-AS <400> 26 aagcttttag tcgtgcttga gagt 24 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-XhoI <400> 27 catatgactc cactcggacc tg 22 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-AS <400> 28 aagctttcat ggctgtgcaa g 21 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-XhoI <400> 29 ctcgaggatg acgatgacaa gtctgtattt ga 32 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-AS <400> 30 aagcttctat cacttaacat c 21 <210> 31 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> EDA-S-H6 <400> 31 catatgcacc atcaccatca ccataaaaac tggaaaacaa gtgca 45 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-S-D4K <400> 32 ctcgaggatg acgatgacaa gagctcccag attcgt 36 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-D4K <400> 33 ctcgaggacg atgacgataa aacccccctg ggccctgcc 39 <210> 34 <211> 639 <212> DNA <213> Escherichia coli EDA <400> 34 atgaaaaact ggaaaacaag tgcagaatca atcctgacca ccggcccggt tgtaccggtt 60 atcgtggtaa aaaaactgga acacgcagtg ccgatggcaa aagcgttggt tgctggtggg 120 gtgcgcgttc tggaagtgac tctgcgtacc gagtgtgcag ttgacgctat ccgtgctatc 180 gccaaagaag tgcctgaagc gattgtgggt gccggtacgg tgctgaatcc acagcagctg 240 gcagaagtca ctgaagcggg tgcacagttc gcaattagcc cgggtctgac cgagccgctg 300 ctgaaagctg ctaccgaagg gactattcct ctaattccgg ggatcagcac tgtttccgaa 360 ctgatgctgg gtatggacta cggtttgaaa gagttcaaat tcttcccggc tgaagctaac 420 ggcggcgtga aagccctgca ggcgatcgcg ggtccgttct cccaggtccg tttctgcccg 480 acgggtggta tttctccggc taactaccgt gactacctgg cgctgaaaag cgtgctgtgc 540 atcggtggtt cctggctggt tccggcagat gcgctggaag cgggcgatta cgaccgcatt 600 actaagctgg cgcgtgaagc tgtagaaggc gctaagctg 639 <210> 35 <211> 1374 <212> DNA <213> human ppGRN <400> 35 agttccccaa agataacaca gctttgcaca gtggatgttt acttgctggt ggtcttatct 60 aagatcaaca ttggcagctg tgcccggaga ggcctccagg gtccaggtcc aatgcacttc 120 cctctcagaa gaggcatccg ctaaaatagg gaccaaagct gctggaggga ggcaaggcaa 180 gctgctatgt gaaaaaacgc caggccaggc agtcatgtca cacctggcag aaatgactga 240 agcatagcca ctggctgaag ttatccccac acccactctc tggagaggat gatcaggagc 300 agtctgctca accgggaggt gggactcctc ctcgggaagg tgtagaatca ccagcctggc 360 tccctgcgga ctcccggggc tcacagaggc cagagcagca acagcacatg ggaaacaacg 420 gggcgctgga ctggggaggt ctcagagctc tcctagtgat gacagcctca ttttacccag 480 ggagaaaggg cgagtaagct aaggtcacac agcaacaaag ctgcacccag accccagagc 540 cactctcctc cctccctcct ccaccagggc catgcccact tggggcaccc cgccaccgtg 600 ttccagggac agctggagca catgcttctt ccctcgccaa cccagcaatt ccgcagggca 660 tctgacctcc actgttgact tctacccaga ggacaagaac atttttagtt cccaaggaat 720 gtacatcagc cccacggaag ctaggccacc tctgggatgg ggttgctggt ttagaacaaa 780 cgccagtcat cctatataag gacctgacag ccaccaggca ccacctccgc caggaactgc 840 aggcccacct gtctgcaacc cagctgaggc catgccctcc ccagggaccg tctgcagcct 900 cctgctcctc ggcatgctct ggctggactt ggccatggca ggctccagct tcctgagccc 960 tgaacaccag agagtccagc agagaaagga gtcgaagaag ccaccagcca agctgcagcc 1020 ccgagctcta gcaggctggc tccgcccgga agatggaggt caagcagaag gggcagagga 1080 tgaactggaa gtccggttca acgccccctt tgatgttgga atcaagctgt caggggttca 1140 gtaccagcag cacagccagg ccctggggaa gtttcttcag gacatcctct gggaagaggc 1200 caaagaggcc ccagccgaca agtgatcgcc cacaagcctt actcacctct ctctaagttt 1260 agaagcgctc atctggcttt tcgcttgctt ctgcagcaac tcccacgact gttgtacaag 1320 ctcaggaggc gaataaatgt tcaaactgta aaaaaaaaaa aaaaaaaaaa aaaa 1374 <210> 36 <211> 573 <212> DNA <213> human IL-2 <400> 36 acctcaactc ctgccacaat gtacaggatg caactcctgt cttgcattgc actaagtctt 60 gcacttgtca caaacagtgc acctacttca agttctacaa agaaaacaca gctacaactg 120 gagcatttac tgctggattt acagatgatt ttgaatggaa ttaataatta caagaatccc 180 aaactcacca ggatgctcac atttaagttt tacatgccca agaaggccac agaactgaaa 240 catcttcagt gtctagaaga agaactcaaa cctctggagg aagtgctaaa tttagctcaa 300 agcaaaaact ttcacttaag acccagggac ttaatcagca atatcaacgt aatagttctg 360 gaactaaagg gatctgaaac aacattcatg tgtgaatatg ctgatgagac agcaaccatt 420 gtagaatttc tgaacagatg gattaccttt tgtcaaagca tcatctcaac actgacttga 480 taattaagtg cttcccactt aaaacatatc aggccttcta tttatttaaa tatttaaatt 540 ttatatttat tgttgaatgt atggtttgct acc 573 <210> 37 <211> 180 <212> DNA <213> human mp-INS <400> 37 tttgtcaacc aacatttatg tggatcacat ttagtagagg ctttgtatct tgtttgtggt 60 gaacgtggat ttttctatac acctaagaca cgtagatctc ctaatggaaa acgtggtatt 120 gttgaacaat gctgtacatc aatctgttca ttgtatcaac ttgagaacta ctgtaactaa 180 180 <210> 38 <211> 196 <212> DNA <213> human EGF <400> 38 gatccgtagt tgaaggagtt taatcgatga actctgactc cgaatgcccg ctgtctcacg 60 acggttattg cctgcatgat ggtgtttgta tgtatatcga agctctggac aaatatgctt 120 gcaactgtgt tgttggttac atcggtgagc gttgccagta tcgcgacctg aaatggtggg 180 aactgcgtta atgatc 196 <210> 39 <211> 528 <212> DNA <213> human FTN-L <400> 39 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aattggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 ctcatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 gggctgggcg agtatctctt cgaaaggctc actctcaagc acgactaa 528 <210> 40 <211> 522 <212> DNA <213> human G-CSF <400> 40 acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg cttagagcaa 60 gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctgtgtgc cacctacaag 120 ctgtgccacc ccgaggagct ggtgctgctc ggacactctc tgggcatccc ctgggctccc 180 ctgagcagct gccccagcca ggccctgcag ctggcaggct gcttgagcca actccatagc 240 ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc cgagttgggt 300 cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat ctggcagcag 360 atggaagaac tgggaatggc ccctgccctg cagcccaccc agggtgccat gccggccttc 420 gcctctgctt tccagcgccg ggcaggaggg gtcctagttg cctcccatct gcagagcttc 480 ctggaggtgt cgtaccgcgt tctacgccac cttgcccagc cc 522 <210> 41 <211> 350 <212> DNA <213> ADI <400> 41 atgtctgtgt ttgatagcaa atttaaagga attcacgttt attcagaaat tggtgaatta 60 gaatcagttc tagttcacga accaggacgc gaaattgact atattacacc agctagacta 120 gatgaattat tattctcagc tatcttagaa agccatgatg ctagaaaaga acacaaacaa 180 ttcgtagcag aattaaaagc aaacgacatc aatgttgttg aattaattga tttagttgct 240 gaaacatacg atttagcatc acaagaagct aaagataaat taatcgaaga atttttagaa 300 gactcagaac cagttctatc agaagaacac aaagtagttg taaggaactt 350 <110> Korea University Research and Business Foundation <120> Method for Preparing Recombinant Protein Using EDA as a Fusion Expression Partner <130> P15-B010 <160> 41 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> EDA-S <400> 1 catatgaaaa actggaaaac aagtgca 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EDA-AS <400> 2 ctcgagcagc ttagcgcctt ctac 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-S-NdeI <400> 3 catatgggct ccagcttcct g 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-AS-HindIII <400> 4 aagctttcac ttgtcggct 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-S-NdeI <400> 5 catatggcac ctacttcaag t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> HIL-2-AS-HindIII <400> 6 aagcttttat caagtcagtg t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> Mp-INS-S-NdeII <400> 7 catatgtttg tcaaccaaca t 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-AS-Hin III <400> 8 aagcttttag ttacagtagt tc 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-S-NdeI <400> 9 catatgaact ctgactccga atgc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-AS-HindIII <400> 10 aagcttttaa cgcagttccc acca 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> ≪ 223 > hFTN-L-S-NdeI <400> 11 catatgagct cccagattcg t 21 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> ≪ 223 > hFTN-L-AS-HindIII <400> 12 aagcttttag tcgtgcttga gagt 24 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-NdeI <400> 13 catatgactc cactcggacc tg 22 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-AS-HindIII <400> 14 aagctttcat ggctgtgcaa g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-NdeI <400> 15 catatgtctg tatttgacag t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-AS-HindIII <400> 16 aagcttctat cacttaacat c 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-S-XhoI <400> 17 ctcgagggct ccagcttcct g 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN-AS <400> 18 aagctttcac ttgtcggct 19 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-S-XhoI <400> 19 ctcgaggcac ctacttcaag t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2-AS <400> 20 aagcttttat caagtcagtg t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> Mp-INS-S-XhoI <400> 21 ctcgagtttg tcaaccaaca t 21 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS-AS <400> 22 aagcttttag ttacagtagt tc 22 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-S-XhoI <400> 23 ctcgagaact ctgactccga atgc 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF-AS <400> 24 aagcttttaa cgcagttccc acca 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-S-XhoI <400> 25 catatgagct cccagattcg t 21 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-AS <400> 26 aagcttttag tcgtgcttga gagt 24 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-XhoI <400> 27 catatgactc cactcggacc tg 22 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-AS <400> 28 aagctttcat ggctgtgcaa g 21 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-XhoI <400> 29 ctcgaggatg acgatgacaa gtctgtattt ga 32 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-AS <400> 30 aagcttctat cacttaacat c 21 <210> 31 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> EDA-S-H6 <400> 31 catatgcacc atcaccatca ccataaaaac tggaaaacaa gtgca 45 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L-S-D4K <400> 32 ctcgaggatg acgatgacaa gagctcccag attcgt 36 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF-S-D4K <400> 33 ctcgaggacg atgacgataa aacccccctg ggccctgcc 39 <210> 34 <211> 639 <212> DNA <213> Escherichia coli EDA <400> 34 atgaaaaact ggaaaacaag tgcagaatca atcctgacca ccggcccggt tgtaccggtt 60 atcgtggtaa aaaaactgga acacgcagtg ccgatggcaa aagcgttggt tgctggtggg 120 gtgcgcgttc tggaagtgac tctgcgtacc gagtgtgcag ttgacgctat ccgtgctatc 180 gccaaagaag tgcctgaagc gattgtgggt gccggtacgg tgctgaatcc acagcagctg 240 gcagaagtca ctgaagcggg tgcacagttc gcaattagcc cgggtctgac cgagccgctg 300 ctgaaagctg ctaccgaagg gactattcct ctaattccgg ggatcagcac tgtttccgaa 360 ctgatgctgg gtatggacta cggtttgaaa gagttcaaat tcttcccggc tgaagctaac 420 ggcggcgtga aagccctgca ggcgatcgcg ggtccgttct cccaggtccg tttctgcccg 480 acgggtggta tttctccggc taactaccgt gactacctgg cgctgaaaag cgtgctgtgc 540 atcggtggtt cctggctggt tccggcagat gcgctggaag cgggcgatta cgaccgcatt 600 actaagctgg cgcgtgaagc tgtagaaggc gctaagctg 639 <210> 35 <211> 1374 <212> DNA <213> human ppGRN <400> 35 agttccccaa agataacaca gctttgcaca gtggatgttt acttgctggt ggtcttatct 60 aagatcaaca ttggcagctg tgcccggaga ggcctccagg gtccaggtcc aatgcacttc 120 cctctcagaa gaggcatccg ctaaaatagg gaccaaagct gctggaggga ggcaaggcaa 180 gctgctatgt gaaaaaacgc caggccaggc agtcatgtca cacctggcag aaatgactga 240 agcatagcca ctggctgaag ttatccccac acccactctc tggagaggat gatcaggagc 300 agtctgctgc accgggaggt gggactcctc ctcgggaagg tgtagaatca ccagcctggc 360 tccctgcgga ctcccggggc tcacagaggc cagagcagca acagcacatg ggaaacaacg 420 gggcgctgga ctggggaggt ctcagagctc tcctagtgat gacagcctca ttttacccag 480 ggagaaaggg cgagtaagct aaggtcacac agcaacaaag ctgcacccag accccagagc 540 cactctcctc cctccctcct ccaccagggc catgcccact tggggcaccc cgccaccgtg 600 ttccagggac agctggagca catgcttctt ccctcgccaa cccagcaatt ccgcagggca 660 tctgacctcc actgttgact tctacccaga ggacaagaac atttttagtt cccaaggaat 720 gtacatcagc cccacggaag ctaggccacc tctgggatgg ggttgctggt ttagaacaaa 780 cgccagtcat cctatataag gacctgacag ccaccaggca ccacctccgc caggaactgc 840 aggcccacct gtctgcaacc cagctgaggc catgccctcc ccagggaccg tctgcagcct 900 cctgctcctc ggcatgctct ggctggactt ggccatggca ggctccagct tcctgagccc 960 tgaacaccag agagtccagc agagaaagga gtcgaagaag ccaccagcca agctgcagcc 1020 ccgagctcta gcaggctggc tccgcccgga agatggaggt caagcagaag gggcagagga 1080 tgaactggaa gtccggttca acgccccctt tgatgttgga atcaagctgt caggggttca 1140 gtaccagcag cacagccagg ccctggggaa gtttcttcag gacatcctct gggaagaggc 1200 caaagaggcc ccagccgaca agtgatcgcc cacaagcctt actcacctct ctctaagttt 1260 agaagcgctc atctggcttt tcgcttgctt ctgcagcaac tcccacgact gttgtacaag 1320 ctcaggaggc gaataaatgt tcaaactgta aaaaaaaaaa aaaaaaaaaa aaaa 1374 <210> 36 <211> 573 <212> DNA <213> human IL-2 <400> 36 acctcaactc ctgccacaat gtacaggatg caactcctgt cttgcattgc actaagtctt 60 gcacttgtca caaacagtgc acctacttca agttctacaa agaaaacaca gctacaactg 120 gagcatttac tgctggattt acagatgatt ttgaatggaa ttaataatta caagaatccc 180 aaactcacca ggatgctcac atttaagttt tacatgccca agaaggccac agaactgaaa 240 catcttcagt gtctagaaga agaactcaaa cctctggagg aagtgctaaa tttagctcaa 300 agcaaaaact ttcacttaag acccagggac ttaatcagca atatcaacgt aatagttctg 360 gaactaaagg gatctgaaac aacattcatg tgtgaatatg ctgatgagac agcaaccatt 420 gtagaatttc tgaacagatg gattaccttt tgtcaaagca tcatctcaac actgacttga 480 taattaagtg cttcccactt aaaacatatc aggccttcta tttatttaaa tatttaaatt 540 ttatatttat tgttgaatgt atggtttgct acc 573 <210> 37 <211> 180 <212> DNA <213> human mp-INS <400> 37 tttgtcaacc aacatttatg tggatcacat ttagtagagg ctttgtatct tgtttgtggt 60 gaacgtggat ttttctatac acctaagaca cgtagatctc ctaatggaaa acgtggtatt 120 gttgaacaat gctgtacatc aatctgttca ttgtatcaac ttgagaacta ctgtaactaa 180 180 <210> 38 <211> 196 <212> DNA <213> human EGF <400> 38 gatccgtagt tgaaggagtt taatcgatga actctgactc cgaatgcccg ctgtctcacg 60 acggttattg cctgcatgat ggtgtttgta tgtatatcga agctctggac aaatatgctt 120 gcaactgtgt tgttggttac atcggtgagc gttgccagta tcgcgacctg aaatggtggg 180 aactgcgtta atgatc 196 <210> 39 <211> 528 <212> DNA <213> human FTN-L <400> 39 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aattggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 ctcatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 gggctgggcg agtatctctt cgaaaggctc actctcaagc acgactaa 528 <210> 40 <211> 522 <212> DNA ≪ 213 > human G-CSF <400> 40 acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg cttagagcaa 60 gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctgtgtgc cacctacaag 120 ctgtgccacc ccgaggagct ggtgctgctc ggacactctc tgggcatccc ctgggctccc 180 ctgagcagct gccccagcca ggccctgcag ctggcaggct gcttgagcca actccatagc 240 ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc cgagttgggt 300 cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat ctggcagcag 360 atggaagaac tgggaatggc ccctgccctg cagcccaccc agggtgccat gccggccttc 420 gcctctgctt tccagcgccg ggcaggaggg gtcctagttg cctcccatct gcagagcttc 480 ctggaggtgt cgtaccgcgt tctacgccac cttgcccagc cc 522 <210> 41 <211> 350 <212> DNA <213> ADI <400> 41 atgtctgtgt ttgatagcaa atttaaagga attcacgttt attcagaaat tggtgaatta 60 gaatcagttc tagttcacga accaggacgc gaaattgact atattacacc agctagacta 120 gatgaattat tattctcagc tatcttagaa agccatgatg ctagaaaaga acacaaacaa 180 ttcgtagcag aattaaaagc aaacgacatc aatgttgttg aattaattga tttagttgct 240 gaaacatacg atttagcatc acaagaagct aaagataaat taatcgaaga atttttagaa 300 gactcagaac cagttctatc agaagaacac aaagtagttg taaggaactt 350
Claims (13)
An expression vector for producing a target protein comprising a gene encoding a target protein and an EDA gene represented by SEQ ID NO: 34 as fusion partners.
The expression vector for producing a target protein according to claim 1, wherein the EDA gene is linked to the target protein gene as a fusion partner.
6. The expression vector for producing a target protein according to claim 5, wherein a polynucleotide encoding a protein cleavage enzyme recognition site is linked between the target protein gene and the EDA gene as a fusion partner.
6. The expression vector of claim 5, wherein the EDA gene is operably linked to a polynucleotide encoding a tag for separation and purification as a fusion partner with the target protein gene.
The expression vector for producing a target protein according to claim 1, wherein the target protein is selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cell protein.
A recombinant microorganism into which the expression vector of claim 1 is introduced.
Culturing the recombinant microorganism of claim 9 to induce expression of a target protein, and recovering the target protein.
12. The method of claim 11, further comprising the step of removing EDA from the recombinant protein.
12. A recombinant protein produced by the method of claim 11, wherein the EDA and the target protein are fused.
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