KR100961528B1 - Method for Over-expressing Human Epidermal Growth Factor as Bioactive Form in Escherichia. coli - Google Patents
Method for Over-expressing Human Epidermal Growth Factor as Bioactive Form in Escherichia. coli Download PDFInfo
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- KR100961528B1 KR100961528B1 KR1020070104748A KR20070104748A KR100961528B1 KR 100961528 B1 KR100961528 B1 KR 100961528B1 KR 1020070104748 A KR1020070104748 A KR 1020070104748A KR 20070104748 A KR20070104748 A KR 20070104748A KR 100961528 B1 KR100961528 B1 KR 100961528B1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 대장균에서 활성형 인간 상피세포 성장인자를 대량 생산하기 위하여 인간 상피세포 성장인자와 이의 N-말단 또는 C-말단에 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질이 융합된 융합 단백질을 코딩하는 핵산분자가 조절서열에 작동가능하게 연결되도록 포함된 재조합 발현벡터, 상기 재조합 발현벡터로 형질전환된 대장균 숙주세포, 상기 대장균 숙주세포를 배양하여 상기 융합 단백질을 발현시키고 분리함으로써 활성형 인간 상피세포 성장인자를 대량 제조하는 방법에 관한 것이다.The present invention provides a mass of human epidermal growth factor and its fragment at the N-terminus or C-terminus, maltose binding protein, disulfide binding A protein, disulfide binding C protein for mass production of active human epidermal growth factor in E. coli. And a recombinant expression vector comprising a nucleic acid molecule encoding a fusion protein to which a peptide or protein selected from the group consisting of a signal sequence of pectate lyase B is operably linked to a regulatory sequence, transformed with the recombinant expression vector. The present invention relates to a method for mass production of an active human epidermal growth factor by culturing E. coli host cells and E. coli host cells to express and isolate the fusion protein.
인간 상피세포 성장인자, 시뉴클레인, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열 Signal Sequences of Human Epidermal Growth Factor, Synuclein, Maltose Binding Protein, Disulfide Binding A Protein, Disulfide Binding C Protein, and Pectate Lyase B
Description
본 발명은 활성형 인간 상피세포 성장인자의 대량 제조방법에 관한 것으로서, 보다 자세하게는 인간 상피세포 성장인자와 이의 N-말단 또는 C-말단에 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질이 융합된 융합 단백질을 코딩하는 핵산분자가 조절서열에 작동가능하게 연결되도록 포함된 재조합 발현벡터, 상기 재조합 발현벡터로 형질전환된 대장균 숙주세포, 상기 대장균 숙주세포를 배양하여 상기 융합 단백질을 발현시키고 분리함으로써 활성형 인간 상피세포 성장인자를 대량 제조하는 방법에 관한 것이다.The present invention relates to a method for mass-producing active human epidermal growth factor, and more particularly, to human epidermal growth factor and its N-terminus or C-terminus, fragments of synuclein, maltose binding protein, disulfide binding A protein, A recombinant expression vector comprising a nucleic acid molecule encoding a fusion protein fused to a peptide or protein selected from the group consisting of a disulfide binding C protein and a signal sequence of pectate lyase B, the recombinant expression vector The present invention relates to a method for mass-producing an active human epidermal growth factor by culturing E. coli host cells transformed with E. coli host cells, expressing and isolating the fusion protein.
상피세포 성장인자 (epidermal growth factor, EGF)는 3개의 디설파이드 결합 (disulfide bond)을 갖는 단일 단백질로서 1962년 쥐의 악하선 (submaxillary gland)에서 신경 성장인자 (nerve growth factor)를 분리하던 중에 발견되었고 (Cohen, S. J. Biol Chem. 1962, vol.237, p.1555), Savage에 의해 그 일차 구조가 밝혀졌다 (Savage, C. R. et. al. J. Biol Chem. 1972, vol.247, p.7609). 1975년에 Cohen과 Carpenter는 사람의 뇨에서 최초로 인간 상피세포 성장인자 (human epidermal growth factor)를 분리 · 정제하였다 (Cohen, S. et. al. Adv. Metab. Disord. 1975, vol.8, p.265).Epidermal growth factor (EGF), a single protein with three disulfide bonds, was discovered in 1962 when the nerve growth factor was isolated from the submaxillary gland of rats ( Cohen, SJ Biol Chem. 1962, vol. 237, p. 1555), and its primary structure was revealed by Savage (Savage, CR et. Al. J. Biol Chem. 1972, vol. 247, p. 7609). In 1975, Cohen and Carpenter first isolated and purified human epidermal growth factor from human urine (Cohen, S. et. Al. Adv. Metab. Disord. 1975, vol. 8, p. .265).
인간 상피세포 성장인자는 53개의 아미노산 (159개의 뉴클레오티드)으로 구성된 단백질로서, 전체 분자량이 6,300 달톤 (6.3 kDa)에 이른다. 47번째에 위치한 류신 (leucine) 부분은 인간 상피세포 성장인자의 생체내 활성에 필수적인 역할을 담당하며, 이를 발린 (valine) 또는 이소류신 (isoleucin)으로 치환하할 경우에는 생물학적 활성이 감소하며, 37번째에 위치한 티로신 (tyrosine)은 베타 구조의 수소결합에 중요한 역할을 담당하는 것으로 알려져 있다.Human epidermal growth factor is a protein consisting of 53 amino acids (159 nucleotides) and has a total molecular weight of 6,300 Daltons (6.3 kDa). The leucine region, located at 47, plays an essential role in the in vivo activity of human epidermal growth factor, and when replaced with valine or isoleucine, the biological activity decreases. Tyrosine is known to play an important role in hydrogen bonding of the beta structure.
현재 인간 상피세포 성장인자와 같은 인간 유래 재조합 단백질은 다양한 형질전환 미생물들을 이용하여 제조되고 있다. 이러한 형질전환 미생물 중 대장균을 이용할 경우에는 여러 가지 측면에서 장점이 있다. 첫째로 단위 세포당 목적단백질의 발현율이 다른 미생물들에 비해 월등히 높다는 점이며, 둘째로 유전적인 특성이 많이 밝혀져 있어 다양한 유전공학적 시도를 할 수 있다는 것이며, 셋째로 대량 생산을 용이하게 진행할 수 있다는 점이다.Currently, human-derived recombinant proteins such as human epidermal growth factor have been produced using a variety of transgenic microorganisms. The use of Escherichia coli among these transforming microorganisms has advantages in several aspects. First, the expression rate of the target protein per unit cell is much higher than that of other microorganisms. Second, many genetic characteristics are revealed, and various genetic engineering attempts can be made. Third, mass production can be easily performed. to be.
대장균에서 재조합 단백질의 신호서열을 이용하여 분비시킬 경우 단백질의 올바른 접힘 (folding)을 유도하여 불용성 단백질 응집체 (inclusion body)의 형성을 방지할 수 있고, 분비 과정을 통해 N-단편의 분비 신호서열이 제거되어 자연 상 태에 존재하는 것과 동일한 아미노산 서열을 유지할 수 있는 장점을 지니고 있다. 한편, 융합 단백질을 이용하여 발현시킬 경우 올바른 접힘을 유도하여 수용성 상태로 발현될 수 있도록 도와주는 장점을 지니고 있다.When E. coli is secreted using the signal sequence of the recombinant protein, it is possible to prevent the formation of insoluble protein aggregates by inducing the correct folding of the protein, and the secretion signal sequence of the N-fragment is secreted through the secretion process. It has the advantage of being removed to maintain the same amino acid sequence as it is in nature. On the other hand, when expressed using a fusion protein has the advantage of helping to be expressed in a water-soluble state by inducing the correct folding.
대장균을 이용한 인간 상피세포 성장인자 발현에 대하여 다양한 연구들이 진행되어 왔는데, 그 중 신호서열 펩타이드를 이용한 융합 방법이 많이 시도되었다. Qin 등은 세포외막 단백질 A (OmpA) 분비 신호서열을 이용하여 인간 상피세포 성장인자를 발현시킨 결과 발현 효율이 개선되었다고 보고하였으며(Qin et al., EMT. 2006. p.1-8), 이와 같이 신호서열인 세포외막 단백질 A를 이용하여 배지로 분비하는 기술이 미국특허 제 5,646,015호에 개시되어 있다. 그러나, 전체적인 발현양이 적었으며, 세포질 밖으로의 분비량은 1.1 μg/L로 현저히 적어서 유용하지 않다. 대한민국특허 제 10-0330203호는 안지오게닌을 융합하여 인간 상피세포 성장인자를 발현하는 방법을 제안하였으나, 대부분 불용성 단백질 응집체 형태로 발현되었으며 그 양이 0.5 g/L로 발현양이 적었다.Various studies have been conducted on the expression of human epidermal growth factor using Escherichia coli, among which fusion methods using signal sequence peptides have been tried. Qin et al . Reported that expression of human epidermal growth factor was improved by using extracellular membrane protein A (OmpA) secretion signal sequence (Qin et al ., EMT. 2006. p. 1-8). As described above, US Pat. No. 5,646,015 discloses a technique for secreting into the medium using extracellular membrane protein A, which is a signal sequence. However, the overall expression was low and the secretion out of the cytoplasm was 1.1 μg / L, which is not useful. Korean Patent No. 10-0330203 proposed a method for expressing human epidermal growth factor by fusing angiogenin, but most of them were expressed in the form of insoluble protein aggregates and the amount was less than 0.5 g / L.
대장균을 이용한 인간 상피세포 성장인자의 발현과 관련된 연구로서 베타 락타메이즈의 신호서열을 융합하여 세포질 및 외질에서의 발현에 대한 보고가 있었으며 (Mina et al. Iranian Biomedical Journal, 2004, vol.2, pp.51-61), 알칼라인 포스페테아제의 신호서열을 융합하여 분비를 유도한 연구 (Takanorri et al. Proc. Natl. Acad. Sci., 1985, vol.82, pp.7212-7216) 등이 발표되었다. 이와 같이 대장균에서 인간 상피세포 성장인자의 과발현을 위한 다양한 방법들이 개발되었으나 신호서열을 사용할 경우 활성형으로의 발현 유도가 현저하게 증가하였으나, 불용성 응집체 형태로의 발현이 상당하고, 전체적인 발현양이 적다는 문제점을 지니고 있다. 한편, 효모를 사용한 인간 상피세포 성장인자의 제조방법에 대한 특허로는 인체표피 성장인자의 유전자를 갖는 벡터 및 이를 이용하여 인체표피 성장인자를 생산하는 대한민국특허 제 10-0295909호가 있다. 상기 특허는 메탄자화효모인 한세눌라 폴리모르파를 이용하여 인간 상피세포 성장인자를 발현시키고 이를 배지내로 효율적으로 분비시키는 방법을 개시하고 있는데, 인간 상피세포 성장인자를 효과적으로 발현시켰고 이를 세포외로 분비하여 최대 24 mg/L의 발현양을 나타내었다. 그러나, 효모에서 인간 상피세포 성장인자와 같이 작은 단백질의 과발현시 문제점은 분비된 단백질이 다양한 프로테아제에 의해 분해될 수 있다는 단점과 효모 유래의 당쇄화로 인하여 인체에 적합하지 않다는 문제가 발생할 수 있다.As a study related to the expression of human epidermal growth factor using E. coli, there has been a report on the expression in the cytoplasm and in the extracellular matrix by fusion of beta lactamase signal sequence (Mina et al. Iranian Biomedical Journal, 2004, vol. 2, pp. 51-61), a study that induces secretion by fusing the signal sequence of alkaline phosphate (Takanorri et al. Proc. Natl. Acad. Sci., 1985, vol. 82, pp.7212-7216). Was released. As described above, various methods for overexpression of human epidermal growth factor were developed in Escherichia coli, but the expression of the active sequence was significantly increased when the signal sequence was used, but the expression in the form of insoluble aggregates was considerable and the overall expression amount was low. Has a problem. On the other hand, the patent for a method for manufacturing human epidermal growth factor using yeast is a vector having a gene of human epidermal growth factor and Korean Patent No. 10-0295909 for producing human epidermal growth factor using the same. The patent discloses a method for expressing human epidermal growth factor and efficiently secreting it into a medium using Hansenula polymorpha, a methanated yeast, which effectively expresses human epidermal growth factor and secretes it extracellularly. Expression levels up to 24 mg / L were shown. However, problems in overexpression of small proteins, such as human epidermal growth factor in yeast, may cause disadvantages that secreted proteins may be degraded by various proteases and may not be suitable for the human body due to yeast-derived glycosylation.
이에 본 발명자들은 대장균 숙주세포에서 활성형 인간 상피세포 성장인자를 대량 제조하기 위하여 인간 상피세포 성장인자 단백질을 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질과 융합시켜 발현시킨 결과 인간 상피세포 성장인자 단백질을 활성형으로 과발현시킬 수 있다는 것을 확인하고 본 발명을 완성하기에 이르렀다.Therefore, the present inventors have used human epidermal growth factor protein to produce a large amount of active human epidermal growth factor in E. coli host cells, fragments of synuclein, maltose binding protein, disulfide binding A protein, disulfide binding C protein and pectase lyase. As a result of fusion with a peptide or protein selected from the group consisting of the signal sequence of B, the human epidermal growth factor protein was overexpressed in an active form, and the present invention was completed.
본 발명은 한 관점으로서 인간 상피세포 성장인자와 이의 N-말단 또는 C-말단에 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질이 융합된 융합 단백질을 코딩하는 핵산분자가 조절서열에 작동가능하게 연결되도록 포함된 재조합 발현벡터를 제공한다.In one aspect, the present invention provides a human epithelial growth factor and a signal sequence of a fragment of synuclein, maltose binding protein, disulfide binding A protein, disulfide binding C protein and pectase lyase B at its N-terminus or C-terminus. Provided is a recombinant expression vector comprising a nucleic acid molecule encoding a fusion protein fused to a peptide or protein selected from the group to be operably linked to regulatory sequences.
"인간 상피세포 성장인자"는 159개의 뉴클레오타이드로 이루어져 있으며, 대장균에서 발현되지 않는 3개의 희귀 코돈, 즉 7번째 프롤린(proline)을 코딩하는 코돈 CCC 및 41번째와 45번째 아르기닌(arginine)을 코딩하는 코돈 CGA를 갖는다. 따라서 대장균을 이용하여 인간 상피세포 성장인자를 발현시킬 때에는 상기 희귀 코돈 CCC와 CGA를 대장균이 일반적으로 인식할 수 있는 코돈 즉, CCG/CCT와 CGT/CGC로 각각 치환하거나, 코돈 CCC 또는 CGA를 인식하는 tRNA가 삽입된 균주 [(예: Rosetta (Invitrogen, 미국)]를 사용한다."Human epithelial growth factor" consists of 159 nucleotides and encodes three rare codons that are not expressed in E. coli, the codon CCC encoding the seventh proline and the 41st and 45th arginine Have a codon CGA. Therefore, when expressing human epidermal growth factor using E. coli, the rare codons CCC and CGA are replaced with codons that can be generally recognized by E. coli, that is, CCG / CCT and CGT / CGC, or codon CCC or CGA is recognized. Strain using tRNA inserted (eg, Rosetta (Invitrogen, USA)).
본 발명에서 상기 인간 상피세포 성장인자는 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질 (또는 "융합 파트너"라고 함)에 의하여 활성형 형태로 과발현될 수 있다. 이하, 상기 융합 파트너에 대하여 구체적으로 설명한다. In the present invention, the human epidermal growth factor is a peptide or protein selected from the group consisting of fragments of synuclein, maltose binding protein, disulfide binding A protein, disulfide binding C protein and pectase lyase B (or "fusion partner"). May be overexpressed in the active form. Hereinafter, the fusion partner will be described in detail.
"시뉴클레인의 단편"은 시뉴클레인 단백질 (Synuclein, ATS; S.M. Park et al., Protein Eng. Desl. Sel., 2004, vol.17, pp.251-260)의 119번째 내지 140번째에 위치한 아미노산으로 이루어진 펩타이드를 의미한다. 본 발명에서 시뉴클레인 단편을 코딩하는 핵산분자의 한 양태는 서열번호: 2에 나타낸 염기서열로 이루어진다. 상기 시뉴클레인의 단편이 융합된 인간 상피세포 성장인자를 코딩하는 DNA는 서열번호: 10으로 구성된 프라이머 (ATSF) 및 서열번호: 9로 구성된 프라이머 (EGFR)를 이용하여 PCR을 진행함으로써 제조할 수 있다."Fragments of synuclein" are amino acids located from 119th to 140th of the synuclein protein (Synuclein, ATS; SM Park et al., Protein Eng. Desl. Sel., 2004, vol. 17, pp.251-260) It means a peptide consisting of. One embodiment of the nucleic acid molecule encoding the synuclein fragment in the present invention consists of the nucleotide sequence shown in SEQ ID NO: 2. The DNA encoding the human epidermal growth factor fused to the fragment of the nucleolein can be prepared by PCR using a primer consisting of SEQ ID NO: 10 (ATSF) and a primer consisting of SEQ ID NO: 9 (EGFR). .
"말토오스 결합 단백질 (Maltose Binding Protein, MBP; Genebank의 EF431917)"은 말토덱스트린 (maltodextrin)을 이동시키고 분해하는데 관여하는 단백질로 공지되어 있다. 본 발명에서 말토오스 결합 단백질을 코딩하는 핵산분자의 한 양태는 서열번호: 3에 나타낸 염기서열로 이루어진다. 상기 말토오스 결합 단백질을 코딩하는 핵산분자는 그러한 핵산분자를 포함하는 핵산분자를 주형으로 하고 서열번호: 11로 구성된 프라이머 (MBPF) 및 서열번호: 12로 구성된 프라이머 (MBPR)를 이용하여 PCR을 진행함으로써 제조할 수 있다."Maltose Binding Protein (MBP; EF431917 from Genebank)" is known as a protein involved in the transfer and degradation of maltodextrin. One embodiment of the nucleic acid molecule encoding the maltose binding protein in the present invention consists of the nucleotide sequence shown in SEQ ID NO: 3. The nucleic acid molecule encoding the maltose binding protein is a nucleic acid molecule containing such a nucleic acid molecule as a template and by PCR using a primer consisting of SEQ ID NO: 11 (MBPF) and a primer consisting of SEQ ID NO: 12 (MBPR) It can manufacture.
"디설파이드 결합 A (Disulfide Bond A, DsbA; Genebank의 X80762) 단백질"은 세포외질에서 단백질의 디설파이드 결합, 즉 접힘 (folding)에 관여하는 단백질 중 한 유형으로 공지되어 있다. 본 발명에서 디설파이드 결합 A 단백질을 코딩하는 핵산분자의 한 양태는 서열번호: 4에 나타낸 염기서열로 이루어진다. 상기 디설파이드 결합 A 단백질을 코딩하는 핵산분자는 그러한 핵산분자를 포함하는 핵산분자를 주형으로 하고 서열번호: 13으로 구성된 프라이머 (DsbAF) 및 서열번호: 14로 구성된 프라이머 (DsbAR)를 이용하여 PCR을 진행함으로써 제조할 수 있다."Disulfide Bond A (DsbA; X80762 from Genebank) protein" is known as one type of protein involved in disulfide bonds, ie folding, of proteins in the extracellular matrix. One embodiment of the nucleic acid molecule encoding the disulfide binding A protein in the present invention consists of the nucleotide sequence shown in SEQ ID NO: 4. The nucleic acid molecule encoding the disulfide binding A protein is subjected to PCR using a nucleic acid molecule containing such nucleic acid molecule as a template and a primer consisting of SEQ ID NO: 13 (DsbAF) and a primer consisting of SEQ ID NO: 14 (DsbAR). It can manufacture by doing.
"디설파이드 결합 C (Disulfide Bond C, DsbC; Genebank의 AP009048) 단백질"은 세포외질에서 단백질의 디설파이드 결합, 즉 접힘 (folding)에 관여하는 단백질 중 한 유형으로 공지되어 있다. 본 발명에서 디설파이드 결합 C 단백질을 코딩하는 핵산분자의 한 양태는 서열번호: 5에 나타낸 염기서열로 이루어진다. 상기 디설파이드 결합 C 단백질을 코딩하는 핵산분자는 그러한 핵산분자를 포함하는 핵산분자를 주형으로 하고 서열번호: 15로 구성된 프라이머 (DsbCF) 및 서열번호: 16으로 구성된 프라이머 (DsbCR)를 이용하여 PCR을 진행함으로써 제조할 수 있다."Disulfide Bond C (DsbC; AP009048) protein from Genebank" is known as one type of protein involved in disulfide bonds, ie folding, of proteins in the extracellular matrix. One embodiment of the nucleic acid molecule encoding the disulfide binding C protein in the present invention consists of the nucleotide sequence shown in SEQ ID NO: 5. The nucleic acid molecule encoding the disulfide binding C protein is subjected to PCR using a nucleic acid molecule containing such nucleic acid molecule as a template and a primer consisting of SEQ ID NO: 15 (DsbCF) and a primer consisting of SEQ ID NO: 16 (DsbCR). It can manufacture by doing.
"펙테이트 라이아제 B (Pectate lyase B from Erwina carotovora , PelB; Genebank의 AY585869)"의 신호서열은 발현된 단백질을 세포외질 (periplasm)로 이동 (translocation)시키는데 관여하는 펩타이드로 공지되어 있다. 특히, 상업적으로 입수 가능한 Novagen사의 pET22에 포함되어 있어 상기 벡터를 이용하면 펙테이트 라이아제 B의 신호서열과 인간 상피세포 성장인자로 이루어진 융합 단백질을 코딩하는 핵산분자가 포함된 재조합 발현벡터를 용이하게 제조할 수 있다. 본 발명 에서 펙테이트 라이아제 B 신호서열의 한 양태는 서열번호: 6에 나타낸 염기서열로 이루어진다.Pectate lyase B from Erwina carotovora , PelB; Genebank AY585869) "signal sequence is known as a peptide involved in the translocation of the expressed protein to the periplasm. Particularly, it is contained in the commercially available pET22 of Novagen. A recombinant expression vector comprising a nucleic acid molecule encoding a fusion protein consisting of a signal sequence of pectate lyase B and a human epidermal growth factor can be easily produced in one embodiment of the pectate lyase B signal sequence. Consists of the nucleotide sequence shown in SEQ ID NO: 6.
본 발명의 재조합 발현벡터에 있어서, 인간 상피세포 성장인자와 융합 파트너 사이에 링커 (linker)로서 프로테아제 (protease)에 의해 인식될 수 있는 아미노산 서열을 포함한 펩타이드 또는 단백질을 위치시키는 것이 바람직하다. 본 발명에서는 인간 상피세포 성장인자가 융합 파트너와 융합된 형태로 발현되는데, 상기 링커는 인간 상피세포 성장인자로부터 융합 파트너를 용이하게 분리하기 위하여 삽입한다. 링커로는 예를 들면 엔테로키나아제 (enterokinase)의 DDDDK 또는 팩터 Xa (Factor Xa)의 IEGR과 같은 아미노산 서열을 포함하는 펩타이드, 유비퀴틴 프로테아제가 인식하는 유비퀴틴 (ubiquitine) 및 인테인 (intein)과 같이 자가인식 프로테아제를 이용할 수 있다.In the recombinant expression vector of the present invention, it is preferable to position a peptide or protein comprising an amino acid sequence that can be recognized by a protease as a linker between the human epidermal growth factor and the fusion partner. In the present invention, the human epidermal growth factor is expressed in a fused form with the fusion partner, and the linker is inserted to easily separate the fusion partner from the human epidermal growth factor. Linkers include, for example, peptides containing amino acid sequences such as DDDDK of enterokinase or IEGR of Factor Xa, self-recognition such as ubiquitine and intein recognized by ubiquitin proteases. Proteases can be used.
한편, 본 발명에서 "조절서열"은 인간 상피세포 성장인자와 융합 파트너로 이루어진 융합 단백질의 발현에 필수적이거나 이로운 핵산서열들을 의미한다. 그러한 조절서열에는 이에 제한되는 것은 아니지만, 신호 분비서열, 업스트림(upstream) 활성화 서열 또는 인핸서(enhancer), 폴리아데닐화 서열, 프로펩타이드(propeptide) 서열 및 전사 종결인자 등을 포함한다. 본 발명에서 조절서열은 최소한 프로모터를 포함하여야 한다. 본 발명에서는 대장균에서 프로모터로서 작용할 수 있는 모든 프로모터를 이용하는 것이 가능하지만, T7 프로모터를 이용하는 것이 특히 바람직하다. 다른 조절서열들은 융합 단백질의 발현양을 더욱 증가시키기 위해서 선택적으로 포함될 수 있다.Meanwhile, in the present invention, "regulatory sequence" means nucleic acid sequences essential or beneficial for expression of a fusion protein composed of human epidermal growth factor and a fusion partner. Such regulatory sequences include, but are not limited to, signal secretion sequences, upstream activation sequences or enhancers, polyadenylation sequences, propeptide sequences, transcription terminators, and the like. In the present invention, the regulatory sequence should include at least a promoter. In the present invention, it is possible to use all promoters that can act as promoters in E. coli, but it is particularly preferable to use the T7 promoter. Other regulatory sequences can optionally be included to further increase the expression level of the fusion protein.
본 발명에서 인간 상피세포 성장인자와 융합 파트너로 이루어진 융합 단백질을 코딩하는 핵산분자는 프로모터를 포함한 상기 조절서열에 작동가능하게 연결되어야 대장균 숙주세포에서 발현될 수 있다. 용어 "작동가능하게 연결된"이란 한 핵산서열이 다른 핵산서열과 기능적 관계로 배치된 상태를 의미한다. 예를 들면, 신호 분비서열이 성숙한 단백질의 분비에 참여함으로써 기능을 발휘했다면 그 단백질에 작동가능하게 연결된 것이다. 프로모터가 코딩 서열의 전사를 조절했다면 그 서열에 작동가능하게 연결된 것이다. 리보좀 결합 자리가 코딩 서열의 해독이 가능한 위치에 놓여져 있다면 그 서열에 작동가능하게 연결된 것이다.In the present invention, a nucleic acid molecule encoding a fusion protein consisting of a human epidermal growth factor and a fusion partner may be operably linked to the regulatory sequence including a promoter to be expressed in E. coli host cells. The term “operably linked” means a state where one nucleic acid sequence is placed in a functional relationship with another nucleic acid sequence. For example, if a signal secretion sequence functions by participating in the secretion of a mature protein, it is operably linked to that protein. If the promoter has regulated the transcription of the coding sequence, it is operably linked to that sequence. If the ribosomal binding site is in a position where translation of the coding sequence is possible, it is operably linked to that sequence.
본 발명은 다른 관점으로서 본 발명의 재조합 발현벡터로 형질전환된 대장균 숙주세포를 제공한다.In another aspect, the present invention provides E. coli host cells transformed with the recombinant expression vector of the present invention.
형질전환은 Davis et al. Basic Methods in Molecular Biology (1986) 및 Sambrook et al. Basic Methods in Molecular Biology와 같은 기본적인 실험 지침서에 기술된 방법에 의해서 실시될 수 있다. 본 발명의 재조합 발현벡터를 대장균 숙주세포로 도입하기 위해서는 대장균 세포막을 수용성(competent)으로 만들어야 한다. 이러한 방법은 대장균을 염화칼슘 용액 또는 폴리에틸렌 글리콜 (polyethylene glycol)과 염화마그네슘이 혼합된 용액에 노출시키는 것을 포함한다. 또한 대장균 세포막은 전기천공에 의해서도 수용성이 될 수 있으며, 상기 전기 전류는 DNA가 세포내로 유입되도록 세포막을 파열시키는 역할을 한다.Transformation is described by Davis et al. Basic Methods in Molecular Biology (1986) and Sambrook et al. It may be carried out by a method described in basic experimental guidelines such as Basic Methods in Molecular Biology. In order to introduce the recombinant expression vector of the present invention into E. coli host cells, the E. coli cell membrane must be made soluble. This method involves exposing E. coli to a solution of calcium chloride or a mixture of polyethylene glycol and magnesium chloride. In addition, the E. coli cell membrane may be water-soluble by electroporation, and the electric current serves to rupture the cell membrane so that DNA is introduced into the cell.
본 발명은 또 다른 관점으로서 (1) 본 발명의 재조합 발현벡터로 형질전환된 대장균 숙주세포를 배양하여 융합 단백질을 발현시키는 단계 및 (2) 배양물로부터 상기 융합 단백질을 분리하는 단계를 포함한 활성형 인간 상피세포 성장인자를 대량으로 제조하는 방법을 제공한다.In another aspect, the present invention provides an active type comprising the steps of: (1) culturing E. coli host cells transformed with the recombinant expression vector of the present invention to express a fusion protein; and (2) separating the fusion protein from the culture. Provided are methods for producing human epidermal growth factor in large quantities.
본 발명에서 대장균은 공지된 기술을 이용해서 인간 상피세포 성장인자와 융합 파트너로 이루어진 융합 단백질의 생산에 적합한 영양 배지 (예, LB 또는 2xYT)에서 배양할 수 있다. 예를 들어, 대장균 숙주세포들은 적합한 배지와 융합 단백질이 발현 및/또는 분리되는 것을 허용하는 조건 하에, 실시된 실험실 또는 산업용 발효기에서 소규모 혹은 대규모 발효, 셰이크 플라스크 배양에 의해서 배양될 수 있다. 배양은 공지된 기술을 사용해서 탄소, 질소 공급원 및 무기염을 포함하는 적합한 영양배지에서 진행한다. 적합한 배지는 상업적인 공급자로부터 입수 가능하고, 예를 들면, American Type Culture Collection의 카탈로그 등에 기재된 성분 및 이들의 조성 비율에 따라 만들 수도 있다.In the present invention, E. coli can be cultured in a nutrient medium (eg, LB or 2xYT) suitable for the production of a fusion protein consisting of human epidermal growth factor and fusion partner using known techniques. For example, E. coli host cells may be cultured by small or large scale fermentation, shake flask culture in a laboratory or industrial fermentor performed under conditions that allow for the expression and / or isolation of the appropriate medium and fusion protein. The cultivation is carried out in a suitable nutrient medium containing carbon, nitrogen sources and inorganic salts using known techniques. Suitable media are available from commercial suppliers and may be made, for example, according to the components and proportions thereof as described in the catalog of the American Type Culture Collection.
본 발명에서는 재조합 발현벡터가 조절서열로서 T7 프로모터를 포함할 경우에 형질전환된 대장균 숙주세포를 배양하던 중에 배양액의 흡광도(600nm)가 0.6~0.8일 때 IPTG (Isoprophyl-β-thiogalactosidase)를 첨가하여 본 발명에 따른 융합 단백질의 발현을 유도하는 것이 바람직하다.In the present invention, when the recombinant expression vector contains a T7 promoter as a regulatory sequence, when the absorbance (600 nm) of the culture medium is 0.6-0.8, the IPTG (Isoprophyl-β-thiogalactosidase) is added by culturing the transformed E. coli host cell. It is preferred to induce the expression of the fusion protein according to the invention.
이러한 배양물로부터 본 발명의 융합 단백질은 당업계에 공지된 방법에 의해 분리될 수 있다. 예를 들어서, 융합 단백질은 이에 제한되는 것은 아니지만, 원심분리, 여과, 추출, 분무 건조, 증발 또는 침전을 포함하는 전통적인 방법에 의해서 배양물로부터 분리할 수 있다. 더 나아가 융합 단백질은 크로마토그래피 (예를 들면, 이온 교환, 친화성, 소수성 및 크기별 배제), 전기영동, 분별용해도 (예를 들면, 암모늄 설페이트 침전) 또는 추출을 포함하여 당업계에 공지된 다양한 방법을 통해서 정제될 수 있다.From such cultures the fusion proteins of the invention can be isolated by methods known in the art. For example, the fusion protein can be separated from the culture by traditional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Further, the fusion protein may be subjected to various methods known in the art, including chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (eg, ammonium sulfate precipitation) or extraction. It can be purified through.
본 발명에 따라 인간 상피세포 성장인자 단백질을 시뉴클레인의 단편, 말토오스 결합 단백질, 디설파이드 결합 A 단백질, 디설파이드 결합 C 단백질 및 펙테이트 라이아제 B의 신호서열로 이루어진 그룹으로부터 선택된 펩타이드 또는 단백질과 융합시켜 발현할 경우에는 상기 인간 상피세포 성장인자 단백질을 활성형 형태로 과발현시킬 수 있다.According to the present invention, the human epidermal growth factor protein is expressed by fusion with a peptide or protein selected from the group consisting of a fragment of synuclein, a maltose binding protein, a disulfide binding A protein, a disulfide binding C protein and a signal sequence of pectate lyase B. In this case, the human epidermal growth factor protein may be overexpressed in an active form.
이하, 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.
실시예 1: 희귀 코돈이 치환된 인간 상피세포 성장인자의 발현Example 1: Expression of human epidermal growth factor substituted with rare codons
대장균 BL21(DE3)에서 인간 상피세포 성장인자를 발현하기 위하여 희귀 코돈을 대장균이 인식할 수 있는 코돈으로 치환한 인간 상피세포 성장인자를 제조하였다. 즉, 인간 상피세포 성장인자의 서열인 서열번호: 1의 염기서열을 참조하여 MEGFF (서열번호: 7) 및 MEGFR (서열번호: 8) 프라이머를 제조하고 서열번호: 1의 인간 상피세포 성장인자를 주형으로 PCR을 수행하여 제조하였다. 상기 PCR 산물을 발현벡터 pET26b(+) (Novagen, 미국)에 도입하여 클로닝한 후 재조합 발현벡터 "pEEGF"를 수득하였다 (도 1).In order to express human epidermal growth factor in Escherichia coli BL21 (DE3), a human epidermal growth factor in which a rare codon was substituted with a codon that E. coli could recognize was prepared. That is, the MEGFF (SEQ ID NO: 7) and the MEGFR (SEQ ID NO: 8) primers were prepared by referring to the nucleotide sequence of SEQ ID NO: 1, which is the sequence of human epidermal growth factor, and the human epidermal growth factor of SEQ ID NO: Prepared by performing PCR with the template. The PCR product was introduced into the expression vector pET26b (+) (Novagen, USA) and cloned to obtain the recombinant expression vector “pEEGF” (FIG. 1).
재조합 발현벡터 pEEGF를 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 아가평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 LB 배지 (Tryptone 10 g/L, Yeast 5 g/L, NaCl 5 g/L)에서 37℃의 조건으로 배양하였다. 재조합 발현벡터 pEEGF로 형질전환된 균주의 배양액의 흡광도(600 nm)가 0.6~0.8일 때 0.2 mM IPTG (Isoprophy-β-thiogalactoside)를 첨가하여 인간 상피세포 성장인자의 발현을 유 도하였다.The recombinant expression vector pEEGF was introduced into E. coli BL21 (DE3) strain and transformed, and the transformed strain was selected from LB agar plate medium to which the antibiotic kanamycin was added. Subsequently, the cells were cultured at 37 ° C. in LB medium (Tryptone 10 g / L, Yeast 5 g / L, NaCl 5 g / L) for expression of human epidermal growth factor. When the absorbance (600 nm) of the culture medium transformed with the recombinant expression vector pEEGF was 0.6-0.8, 0.2 mM IPTG (Isoprophy-β-thiogalactoside) was added to induce the expression of human epidermal growth factor.
배양물로부터 균주를 회수한 다음, 대장균내 인간 상피세포 성장인자를 다음과 같이 회수하였다. 배양액 20 ml을 4℃에서 12,000 rpm으로 10분 동안 원심분리하여 침전물을 수득하고, 이 침전물을 인산완충용액 (pH 7.4)에 현탁하였다. 이어서, 현탁액을 초음파 처리(sonication)하여 현탁액 속의 모든 세포를 파쇄하고, 실온에서 12,000rpm으로 5분 동안 원심분리하여 세포 파편(debris)이 제거된 상층액을 수득하였으며, 이 상층액을 실온에서 12,000rpm으로 10분 동안 원심분리하고, 원심분리후 남은 침전물(하층물)에 증류수를 현탁하여 불용상을 수득하였다.After recovering the strain from the culture, E. coli human epidermal growth factor was recovered as follows. 20 ml of the culture was centrifuged at 12,000 rpm for 10 minutes at 4 ° C. to obtain a precipitate, which was suspended in phosphate buffer (pH 7.4). The suspension was then sonicated to disrupt all the cells in the suspension and centrifuged at 12,000 rpm for 5 minutes at room temperature to give a supernatant free of cell debris, which was 12,000 at room temperature. Centrifugation was performed for 10 minutes at rpm, and distilled water was suspended in the remaining precipitate (lower layer) after centrifugation to obtain an insoluble phase.
앞서 수득한 상층액 및 불용상을 전기영동 및 웨스턴 블롯을 수행한 결과 대부분 불용성 형태로 22.9 mg/L이 생성됨을 알 수 있었다. 한편, 희귀 코돈을 치환하지 않은 경우에도 마찬가지로 모두 비활성형 형태로 발현되었으며 그 농도는 20.6 mg/L였다. 인간 상피세포 성장인자 불용성 형태로 생성된다는 것은 인간 상피세포 성장인자가 비활성형 형태로 발현된다는 것을 의미한다.Electrophoresis and western blot of the supernatant and insoluble phase obtained above showed that 22.9 mg / L was produced in the most insoluble form. On the other hand, even when the rare codon was not substituted, all of them were expressed in an inactive form and the concentration was 20.6 mg / L. Generating in human epidermal growth factor insoluble form means that human epidermal growth factor is expressed in inactive form.
실시예 2: 시뉴클레인의 단편(ATS)이 융합된 인간 상피세포 성장인자의 발현Example 2: Expression of Human Epidermal Growth Factor Fusing Fragment of Synuclein (ATS)
대장균 BL21(DE3)에서 시뉴클레인의 단편(ATS)이 융합된 인간 상피세포 성장인자를 발현하기 위하여 다음과 같이 재조합 발현벡터를 제조하였다. 서열번호: 2의 염기서열을 참조하여 ATSF 프라이머 (서열번호: 10)를 제조하고, 인간 상피세포 성장인자의 서열인 서열번호: 1의 염기서열을 참조하여 EGFR 프라이머 (서열번호: 9)를 제조한 후 상기 프라이머를 이용하여 서열번호: 1의 인간 상피세포 성장인자 를 주형으로 PCR을 수행해서 시뉴클레인의 단편이 융합된 인간 상피세포 성장인자를 코딩하는 DNA를 제조하였다. 상기 PCR 산물을 발현벡터 pET-26b(+)(Novagen, 미국)에 도입하여 클로닝한 후 재조합 발현벡터 "pEATEGF"를 수득하였다.Recombinant expression vectors were prepared as follows to express human epidermal growth factor fused with a fragment of synuclein (ATS) in Escherichia coli BL21 (DE3). An ATSF primer (SEQ ID NO: 10) was prepared by referring to the nucleotide sequence of SEQ ID NO: 2, and an EGFR primer (SEQ ID NO: 9) was prepared by referring to the nucleotide sequence of SEQ ID NO: 1, which is a sequence of human epidermal growth factor. Then, PCR was performed on the human epidermal growth factor of SEQ ID NO: 1 using the primers to prepare DNA encoding human epidermal growth factor in which fragments of the synuclein were fused. The PCR product was introduced into the expression vector pET-26b (+) (Novagen, USA) and cloned to obtain a recombinant expression vector "pEATEGF".
재조합 발현벡터 pEATEGF을 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 아가평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 실시예 1에서와 동일한 방법으로 IPTG 유도를 통하여 인간 상피세포 성장인자의 발현을 유도하였다.Recombinant expression vector pEATEGF was introduced into E. coli BL21 (DE3) strains and transformed, and transformed strains were selected in LB agar plate medium to which the antibiotic kanamycin was added. Then, the expression of human epidermal growth factor was induced through IPTG induction in the same manner as in Example 1 for the expression of human epidermal growth factor.
배양물로부터 균주를 회수한 다음, 대장균내 인간 상피세포 성장인자를 다음과 같이 회수하였다. 배양액 20 ml을 4℃에서 12,000 rpm으로 10분 동안 원심분리하여 침전물을 수득하고, 이 침전물을 인산완충용액 (pH 7.4)에 현탁하였다. 이어서, 현탁액을 초음파 처리하여 현탁액 속의 모든 세포를 파쇄한 후 실온에서 12,000rpm으로 5분 동안 원심분리하여 세포 파편이 제거된 상층액을 수득하였으며, 이 상층액을 실온에서 12,000rpm으로 10분 동안 원심분리하고, 원심분리 후 남은 침전물(하층물)에 증류수를 현탁하여 불용상을 수득하였다.After recovering the strain from the culture, E. coli human epidermal growth factor was recovered as follows. 20 ml of the culture was centrifuged at 12,000 rpm for 10 minutes at 4 ° C. to obtain a precipitate, which was suspended in phosphate buffer (pH 7.4). The suspension was then sonicated to disrupt all cells in the suspension and then centrifuged at 12,000 rpm for 5 minutes at room temperature to obtain a supernatant free of cell debris, which was centrifuged at 12,000 rpm for 10 minutes at room temperature. Separation and distilled water was suspended in the remaining precipitate (lower layer) after centrifugation to obtain an insoluble phase.
앞서 수득한 상층액 및 불용상 각각 20 ㎕ 및 8 ㎕을 전기영동 (도 2) 및 웨스턴 블롯 (도 3)을 수행한 결과, 도 2 및 3에 나타낸 바와 같이 인간 상피세포 성장인자가 대부분 활성형 형태로 31.2 mg/L 생산됨을 확인할 수 있었다 [도 2 및 3에서 M: 단백질 크기 마커, S: 인간 상피세포 성장인자의 표준품 (Peprotech, 미국), 레인 1: 대장균 파쇄 후 총 단백질, 레인 2: 대장균 파쇄 후 활성형 단백질(상등액), 레인 3: 대장균 파쇄 후 침전 단백질(하층물)을 각각 의미함]. 대부분 의 인간 상피세포 성장인자가 상등액에서 검출된다는 것은 활성형 형태로 발현된다는 것을 의미하고, 이는 인간 상피세포 성장인자가 시클로뉴클레인 단편과 융합 발현됨으로써 활성형 형태로 과발현될 수 있다는 것을 의미한다.As a result of electrophoresis (FIG. 2) and Western blot (FIG. 3), 20 μl and 8 μl of the supernatant and insoluble phase, respectively, obtained above, human epidermal growth factor was mostly active as shown in FIGS. 2 and 3. 31.2 mg / L was produced in the form [M: protein size marker, S: standard of human epidermal growth factor (Peprotech, USA), lane 1: total protein after E. coli disruption, lane 2: E. coli crushed active protein (supernatant), Lane 3: E. coli crushed precipitate protein (lower layer) respectively. The detection of most human epidermal growth factor in the supernatant means that it is expressed in the active form, which means that the human epidermal growth factor can be overexpressed in the active form by fusion expression with the cyclonucleic acid fragment.
실시예 3: 말토오스 결합 단백질(MBP)이 융합된 인간 상피세포 성장인자의 발현Example 3: Expression of human epidermal growth factor fused with maltose binding protein (MBP)
대장균 BL21(DE3)에서 말토오스 결합 단백질이 융합된 인간 상피세포 성장인자를 발현하기 위하여 다음과 같이 재조합 발현벡터를 제조하였다. 말토오스 결합 단백질은 서열번호: 3의 염기서열을 참조하여 MBPF (서열번호: 11)와 MBPR 프라이머 (서열번호: 12)를 제조하여 pMAL-p2X(New England Biolab, 영국)를 주형으로 PCR을 수행하여 얻었고, 인간 상피세포 성장인자는 실시예 1과 같이 MEGFF와 MEGFR 프라이머를 이용하여 서열번호: 1의 인간 상피세포 성장인자를 주형으로 PCR을 수행하여 얻었다. 각각의 PCR산물을 발현벡터 pET-26b(+)(Novagen, 미국)에 도입하여 클로닝한 후 재조합 발현벡터 "pEMBEGF"를 수득하였다.In order to express human epidermal growth factor fused to maltose binding protein in Escherichia coli BL21 (DE3), a recombinant expression vector was prepared as follows. Maltose binding protein was prepared by MBPF (SEQ ID NO: 11) and MBPR primer (SEQ ID NO: 12) by PCR using pMAL-p2X (New England Biolab, UK) as a template with reference to the nucleotide sequence of SEQ ID NO: Human epidermal growth factor was obtained by PCR using a human epidermal growth factor of SEQ ID NO: 1 as a template using MEGFF and MEGFR primers as in Example 1. Each PCR product was introduced into the expression vector pET-26b (+) (Novagen, USA) and cloned to obtain a recombinant expression vector "pEMBEGF".
재조합 발현벡터 pEMBEGF을 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 아가평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 실시예 1에서와 동일한 방법으로 IPTG 유도를 통하여 인간 상피세포 성장인자의 발현을 유도하였다.Recombinant expression vector pEMBEGF was introduced into E. coli BL21 (DE3) strains and transformed, and transformed strains were selected from LB agar plate medium to which the antibiotic kanamycin was added. Then, the expression of human epidermal growth factor was induced through IPTG induction in the same manner as in Example 1 for the expression of human epidermal growth factor.
배양물로부터 균주를 회수한 다음, 대장균내 인간 상피세포 성장인자를 다음과 같이 회수하였다. 배양액 20ml을 4℃에서 12,000rpm으로 10분 동안 원심분리하 여 침전물을 수득하고, 완충용액 A [50mM Tris-HCl (pH7.5), 17% Sucrose, 0.5 mM EDTA] 200 ㎕를 넣고 저온에서 15분 동안 반응시킨 후, 완충용액 B [5mM MgSO4] 800 ㎕를 넣고 얼음에서 30분 동안 반응시켰다. 반응 후 상온에서 14,000rpm으로 10분 동안 원심분리하여 상층액을 회수하였고, 상층액은 트리클로로아세트산 (trichloroacetic acid) 농축법을 사용하여 2배 농축하였으며, 침전물은 증류수에 현탁하여 현탁액을 초음파 처리하여 현탁액 속의 모든 세포를 파쇄한 후 실온에서 12,000rpm으로 5분 동안 원심분리하여 세포파편이 제거된 상층액을 수득하였으며, 이 상층액을 실온에서 12,000rpm으로 10분 동안 원심분리하고, 원심분리 후 남은 침전물(하층물)에 증류수를 현탁하여 불용상을 수득하였다.After recovering the strain from the culture, E. coli human epidermal growth factor was recovered as follows. 20 ml of the culture was centrifuged at 12,000 rpm for 10 minutes at 4 ° C. to obtain a precipitate, and 200 µl of buffer A [50 mM Tris-HCl (pH7.5), 17% Sucrose, 0.5 mM EDTA] was added at a low temperature. After reacting for a minute, 800 μl of buffer B [5mM MgSO 4 ] was added thereto and reacted with ice for 30 minutes. After the reaction, the supernatant was recovered by centrifugation at 14,000 rpm for 10 minutes at room temperature. The supernatant was concentrated twice using trichloroacetic acid, and the precipitate was suspended in distilled water to sonicate the suspension. All cells in the suspension were disrupted and then centrifuged at 12,000 rpm for 5 minutes at room temperature to obtain a supernatant free of cell debris, which was centrifuged at 12,000 rpm for 10 minutes at room temperature, and left after centrifugation. Distilled water was suspended in the precipitate (lower layer) to obtain an insoluble phase.
앞서 수득한 상층액 및 불용상 각각 20 ㎕ 및 8 ㎕을 SDS-PAGE 젤 전기영동 및 웨스턴 블롯을 수행한 결과, 도 4 및 도 5에 나타낸 바와 같이 인간 상피세포 성장인자의 92.4%인 65.7 mg/L가 활성형 형태로 생성됨을 알 수 있었고, 그 중 19%인 12.48 mg/L가 세포외질로 분비됨을 알 수 있었다 [도 4 및 5에서 M: 단백질 크기 마커, S: 인간 상피세포 성장인자의 표준, 1: 대장균 파쇄 후 총 단백질, 2: 세포외질내 단백질, 3: 세포내질내 활성형 단백질(상등액), 4: 대장균 파쇄 후 침전 단백질(하층물)]. 대부분의 인간 상피세포 성장인자가 상등액에서 검출된다는 것은 활성형 형태로 발현된다는 것을 의미하고, 이는 인간 상피세포 성장인자가 말토오스 결합 단백질과 융합 발현됨으로써 활성형 형태로 과발현될 수 있다는 것을 의미한다.20 μl and 8 μl of the supernatant and insoluble phase obtained above were subjected to SDS-PAGE gel electrophoresis and Western blot, respectively. As shown in FIGS. 4 and 5, 65.7 mg / of 92.4% of the human epidermal growth factor. It was found that L is produced in the active form, of which 12.48 mg / L was secreted into the extracellular matrix [19] and M: protein size markers and S: human epidermal growth factor. Standard, 1: total protein after E. coli crushing, 2: protein within the extracellular matrix, 3: active protein within the cytoplasm (supernatant), 4: precipitate protein after crushing E. coli (underlayer)]. The detection of most human epidermal growth factor in the supernatant means that it is expressed in the active form, which means that the human epidermal growth factor can be overexpressed in the active form by fusion expression with maltose binding protein.
실시예 4: 디설파이드 결합 A 단백질(DsbA)이 융합된 인간 상피세포 성장인자의 발현Example 4: Expression of human epidermal growth factor fused with disulfide binding A protein (DsbA)
대장균 BL21(DE3)에서 디설파이드 결합 A 단백질이 융합된 인간 상피세포 성장인자를 발현하기 위하여 다음과 같이 재조합 발현벡터를 제조하였다. 디설파이드 결합 A 단백질은 서열번호: 4의 염기서열을 참조하여 DsbAF (서열번호: 13)와 DsbAR 프라이머 (서열번호: 14)를 제작하여 pET-39b(+)(Novagen, 미국)를 주형으로 PCR을 수행하여 얻었고, 인간 상피세포 성장인자는 실시예 1과 같이 MEGFF와 MEGFR 프라이머를 이용하여 서열번호: 1의 인간 상피세포 성장인자를 주형으로 PCR을 수행하여 얻었다. 각각의 PCR산물을 재조합 발현벡터 pET-26b(+)(Novagen, 미국)에 도입하여 클로닝한 후 재조합 발현벡터 "pEDAEGF"를 수득하였다.In order to express a human epidermal growth factor fused with disulfide binding A protein in Escherichia coli BL21 (DE3), a recombinant expression vector was prepared as follows. The disulfide binding A protein was prepared by constructing DsbAF (SEQ ID NO: 13) and DsbAR primer (SEQ ID NO: 14) with reference to the nucleotide sequence of SEQ ID NO: 4, and PCR was performed using pET-39b (+) (Novagen, USA) as a template. The human epidermal growth factor was obtained by PCR using a template of human epidermal growth factor of SEQ ID NO: 1 using MEGFF and MEGFR primers as in Example 1. Each PCR product was introduced into recombinant expression vector pET-26b (+) (Novagen, USA) and cloned to obtain recombinant expression vector “pEDAEGF”.
재조합 발현벡터 pEDAEGF을 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 아가평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 실시예 1에서와 동일한 방법으로 IPTG 유도를 통해 인간 상피세포 성장인자의 발현을 유도하였다.The recombinant expression vector pEDAEGF was introduced into E. coli BL21 (DE3) strain and transformed, and the transformed strain was selected in LB agar plate medium to which the antibiotic kanamycin was added. Then, the expression of human epidermal growth factor was induced through IPTG induction in the same manner as in Example 1 for the expression of human epidermal growth factor.
배양물로부터 균주를 회수한 다음, 대장균내 인간 상피세포 성장인자를 실시예 2와 동일한 방법으로 회수하였다.After recovering the strain from the culture, E. coli human epidermal growth factor was recovered in the same manner as in Example 2.
본 실시예 4에서 발현된 인간 상피세포 성장인자의 90%인 69.0 mg/L가 활성형 형태로 생성됨을 알 수 있었고, 그 중 18.7%인 12.9 mg/L가 외질로 분비됨을 알 수 있었다.It was found that 69.0 mg / L, which is 90% of the human epidermal growth factor expressed in this Example 4, was produced in the active form, of which 12.9 mg / L, which is 18.7%, was secreted into the outer vagina.
실시예 5: 디설파이드 결합 C 단백질(DsbC)이 융합된 인간 상피세포 성장인자의 발현Example 5: Expression of human epidermal growth factor fused with disulfide binding C protein (DsbC)
대장균 BL21(DE3)에서 디설파이드 결합 C 단백질이 융합된 인간 상피세포 성장인자를 발현하기 위하여 다음과 같이 재조합 발현벡터를 제조하였다. 디설파이드 결합 C 단백질은 서열번호: 5를 참조하여 DsbCF (서열번호: 15)와 DsbCR 프라이머 (서열번호: 16)를 제조하여 pET-40b(+)(Novagen, 미국)를 주형으로 PCR을 수행하여 얻었고, 인간 상피세포 성장인자는 실시예: 1과 같이 MEGFF와 MEGFR 프라이머를 이용하여 서열번호: 1의 인간 상피세포성장인자를 주형으로 PCR을 수행하여 얻었다. 각각의 PCR산물을 재조합 발현벡터 pET-26b(+)(Novagen, 미국)에 도입하여 클로닝한 후 재조합 발현벡터 "pEDCEGF"를 수득하였다.In order to express human epidermal growth factor fused with disulfide binding C protein in Escherichia coli BL21 (DE3), a recombinant expression vector was prepared as follows. The disulfide binding C protein was obtained by PCR of pET-40b (+) (Novagen, USA) by preparing DsbCF (SEQ ID NO: 15) and DsbCR primer (SEQ ID NO: 16) with reference to SEQ ID NO: 5. , Human epidermal growth factor was obtained by PCR using a human epidermal growth factor of SEQ ID NO: 1 using the MEGFF and MEGFR primers as in Example 1. Each PCR product was introduced into the recombinant expression vector pET-26b (+) (Novagen, USA) and cloned to obtain a recombinant expression vector "pEDCEGF".
재조합 발현벡터 pEDCEGF을 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 아가평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 실시예 1에서와 동일한 방법으로 IPTG 유도를 통해 인간 상피세포 성장인자의 발현을 유도하였다.The recombinant expression vector pEDCEGF was introduced into E. coli BL21 (DE3) strain and transformed, and the transformed strain was selected from LB agar plate medium to which the antibiotic kanamycin was added. Then, the expression of human epidermal growth factor was induced through IPTG induction in the same manner as in Example 1 for the expression of human epidermal growth factor.
본 실시예 5에서 발현된 인간 상피세포 성장인자의 89%인 60.8 mg/L가 활성형 형태로 생성됨을 알 수 있었고, 그 중 24%인 14.6 mg/L가 세포외질로 분비됨을 알 수 있었다.It was found that 60.8 mg / L, which is 89% of the human epidermal growth factor expressed in Example 5, was produced in the active form, of which 14.6 mg / L, which is 24%, was secreted into the extracellular matrix.
실시예 6: 펙테이트 라이아제 B의 신호서열이 융합된 인간 상피세포 성장인자의 발현 Example 6: Expression of human epidermal growth factor fused to signal sequence of pectate lyase B
대장균 BL21(DE3)에서 펙테이트 라이아제 B의 신호서열이 융합된 인간 상피세포 성장인자를 발현하기 위하여 다음과 같이 재조합 발현벡터를 제조하였다. 펙테이트 라이아제 B의 신호서열로는 pET-22b(+)(Novagen, 미국)의 294-357 뉴클레오타이드 서열인 서열번호: 6을 이용하였다. 상기 pET-22b(+)의 NcoI과 SalI 효소 인식부위에 PEGFF (서열번호: 17)와 MEGFR 프라이머 (서열번호: 8)를 이용하여 서열번호: 1의 인간 상피세포성장인자를 주형으로 PCR을 수행하여 수득한 PCR 산물을 삽입하여 재조합 발현벡터 "pEPBEGF"를 수득하였다. Recombinant expression vectors were prepared as follows to express human epidermal growth factor fused to the signal sequence of pectate lyase B in E. coli BL21 (DE3). As a signal sequence of pectate lyase B, SEQ ID NO: 6, which is a 294-357 nucleotide sequence of pET-22b (+) (Novagen, USA), was used. The human epidermal growth factor of SEQ ID NO: 1 was subjected to PCR using PEGFF (SEQ ID NO: 17) and MEGFR primer (SEQ ID NO: 8) at the NcoI and SalI enzyme recognition sites of pET-22b (+). The obtained PCR product was inserted to obtain a recombinant expression vector "pEPBEGF".
재조합 발현벡터 pEPBEGF를 대장균 BL21(DE3) 균주에 도입하여 형질전환시키고, 항생제 카나마이신이 첨가된 LB 평판배지에서 형질전환된 균주를 선별하였다. 이어서, 인간 상피세포 성장인자의 발현을 위해 실시예 1에서와 동일한 방법으로 IPTG 유도를 통해 인간 상피세포 성장인자의 발현을 유도하였다.The recombinant expression vector pEPBEGF was introduced into E. coli BL21 (DE3) strain and transformed, and the transformed strain was selected in LB plate medium to which the antibiotic kanamycin was added. Then, the expression of human epidermal growth factor was induced through IPTG induction in the same manner as in Example 1 for the expression of human epidermal growth factor.
본 실시예 6에서 발현된 인간 상피세포 성장인자의 81%인 28.6 mg/L가 활성형 형태로 생성됨을 알 수 있었고, 그 중 16%인 4.6 mg/L가 세포외질로 분비됨을 알 수 있었다.It was found that 28.6 mg / L, which is 81% of the human epidermal growth factor expressed in Example 6, was produced in the active form, of which 4.6 mg / L, which is 16%, was secreted into the extracellular matrix.
전술된 실시예 2 내지 6을 통하여 본 발명에 따른 각각의 융합 파트너를 이용하여 발현시킨 인간 상피세포 성장인자의 발현양을 표 2에 나타내었다. Table 2 shows the expression levels of the human epidermal growth factor expressed using the respective fusion partners according to the present invention through Examples 2 to 6 described above.
도 1은 희귀 코돈이 대장균이 인식할 수 있는 코돈으로 치환된 인간 상피세포 성장인자를 발현하기 위한 재조합 발현벡터 pEEGF의 모식도이다.1 is a schematic diagram of a recombinant expression vector pEEGF for expressing a human epidermal growth factor in which a rare codon is substituted with a codon that E. coli can recognize.
도 2는 본 발명에 따라 시뉴클레인의 단편과 융합된 인간 상피세포 성장인자가 활성형으로 발현되는지 여부를 확인하기 위한 전기영동 결과 사진이다.Figure 2 is a photograph of the results of electrophoresis to confirm whether the human epidermal growth factor fused with fragments of the synuclein in accordance with the present invention is expressed in active form.
도 3은 본 발명에 따라 시뉴클레인의 단편과 융합된 인간 상피세포 성장인자가 활성형으로 발현되는지 여부를 확인하기 위한 웨스턴 블롯 결과 사진이다.3 is a photograph of Western blot results for confirming whether human epidermal growth factor fused with fragments of synuclein according to the present invention is expressed in active form.
도 4는 본 발명에 따라 말토오스 결합 단백질과 융합된 인간 상피세포 성장인자가 활성형으로 발현되는지 여부를 확인하기 위한 전이영동 결과 사진이다.Figure 4 is a photogram of the results of metastasis for confirming whether human epidermal growth factor fused with maltose binding protein is expressed as an active form according to the present invention.
도 5는 본 발명에 따라 말토오스 결합 단백질과 융합된 인간 상피세포 성장인자가 활성형으로 발현되는지 여부를 확인하기 위한 웨스턴 블롯 결과 사진이다.5 is a photograph of Western blot results for confirming whether human epidermal growth factor fused with maltose binding protein is expressed as an active form according to the present invention.
<110> Korea Research Institute of Bioscience & Biotechnology <120> Method for Over-expressing Human Epidermal Growth Factor as Bioactive Form in Escherichia.coli <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 159 <212> DNA <213> Homo sapiens <400> 1 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159 <210> 2 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> ATS <400> 2 atgtgcctaa tatntatgac ttcttaaaat gaatgtttct gtactacata attctatntc 60 agagacagt 69 <210> 3 <211> 1101 <212> DNA <213> Artificial Sequence <220> <223> MBP <400> 3 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac t 1101 <210> 4 <211> 627 <212> DNA <213> Artificial Sequence <220> <223> DsbA <400> 4 atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60 cagtatgaag atggtaaaca gtacactacc ctggaaaaac cggtagctgg cgcgccgcaa 120 gtgctggagt ttttctcttt cttctgcccg cactgctatc agtttgaaga agttctgcat 180 atttctgata atgtgaagaa aaaactgccg gaaggcgtga agatgactaa ataccacgtc 240 aacttcatgg gtggtgacct gggcaaagat ctgactcagg catgggctgt ggcgatggcg 300 ctgggcgtgg aagacaaagt gactgttccg ctgtttgaag gcgtacagaa aacccagacc 360 attcgttctg cttctgatat ccgcgatgta tttatcaacg caggtattaa aggtgaagag 420 tacgacgcgg cgtggaacag cttcgtggtg aaatctctgg tcgctcagca ggaaaaagct 480 gcagctgacg tgcaattgcg tggcgttccg gcgatgtttg ttaacggtaa atatcagctg 540 aatccgcagg gtatggatac cagcaatatg gatgtttttg ttcagcagta tgctgataca 600 gtgaaatatc tgtccgagaa aaaataa 627 <210> 5 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> DsbC <400> 5 atgaagaaag gttttatgtt gtttactttg ttagcggcgt tttcaggctt tgctcaggct 60 gatgacgcgg caattcaaca aacgttagcc aaaatgggca tcaaaagcag cgatattcag 120 cccgcgcctg tagctggcat gaagacagtt ctgactaaca gcggcgtgtt gtacatcacc 180 gatgatggta aacatatcat tcaggggcca atgtatgacg ttagtggcac ggctccggtc 240 aatgtcacca ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt 300 tataaagcgc cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac 360 tgccacaaac tgcatgagca aatggcagac tacaacgcgc tggggatcac cgtgcgttat 420 cttgctttcc cgcgccaggg gctggacagc gatgcagaga aagaaatgaa agctatctgg 480 tgtgcgaaag ataaaaacaa agcgtttgat gatgtgatgg caggtaaaag cgtcgcacca 540 gccagttgcg acgtggatat tgccgaccat tacgcacttg gcgtccagct tggcgttagc 600 ggtactccgg cagttgtgct gagcaatggc acacttgttc cgggttacca gccgccgaaa 660 gagatgaaag aattcctcga cgaacaccaa aaaatgacca gcggtaaata a 711 <210> 6 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> PelB <400> 6 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggcc 66 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgataacata tgaatagtga ctctgaatgt ccgg 34 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgataagtcg actcagcgtt cccaccactt caggtcacgg tactgacaac gctc 54 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tgataagtcg actcagcgca gttcccacca 30 <210> 10 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tgataacata tggatcctga caatgaggct tatgaaatgc cttctgagga agggtatcaa 60 gactacgaac ctgaagccaa tagtgactct 90 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgataacata tgaaaataaa aacaggtgca 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgataagaat tcagtctgcg cgtctttcag 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgataacata tgaaaaagat ttggctggcg 30 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tgataagaat tctccttttt tctcgcttaa gta 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tgataacata tgaagaaagg ttttatgttg ttt 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tgataagaat tctttccagc tggtcatttt ttg 33 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tgataaccat ggggaatagt gactctgaat gtccgg 36 <110> Korea Research Institute of Bioscience & Biotechnology <120> Method for Over-expressing Human Epidermal Growth Factor as Bioactive Form in Escherichia.coli <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 159 <212> DNA <213> Homo sapiens <400> 1 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159 <210> 2 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> ATS <400> 2 atgtgcctaa tatntatgac ttcttaaaat gaatgtttct gtactacata attctatntc 60 agagacagt 69 <210> 3 <211> 1101 <212> DNA <213> Artificial Sequence <220> <223> MBP <400> 3 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac t 1101 <210> 4 <211> 627 <212> DNA <213> Artificial Sequence <220> <223> DsbA <400> 4 atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60 cagtatgaag atggtaaaca gtacactacc ctggaaaaac cggtagctgg cgcgccgcaa 120 gtgctggagt ttttctcttt cttctgcccg cactgctatc agtttgaaga agttctgcat 180 atttctgata atgtgaagaa aaaactgccg gaaggcgtga agatgactaa ataccacgtc 240 aacttcatgg gtggtgacct gggcaaagat ctgactcagg catgggctgt ggcgatggcg 300 ctgggcgtgg aagacaaagt gactgttccg ctgtttgaag gcgtacagaa aacccagacc 360 attcgttctg cttctgatat ccgcgatgta tttatcaacg caggtattaa aggtgaagag 420 tacgacgcgg cgtggaacag cttcgtggtg aaatctctgg tcgctcagca ggaaaaagct 480 gcagctgacg tgcaattgcg tggcgttccg gcgatgtttg ttaacggtaa atatcagctg 540 aatccgcagg gtatggatac cagcaatatg gatgtttttg ttcagcagta tgctgataca 600 gtgaaatatc tgtccgagaa aaaataa 627 <210> 5 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> DsbC <400> 5 atgaagaaag gttttatgtt gtttactttg ttagcggcgt tttcaggctt tgctcaggct 60 gatgacgcgg caattcaaca aacgttagcc aaaatgggca tcaaaagcag cgatattcag 120 cccgcgcctg tagctggcat gaagacagtt ctgactaaca gcggcgtgtt gtacatcacc 180 gatgatggta aacatatcat tcaggggcca atgtatgacg ttagtggcac ggctccggtc 240 aatgtcacca ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt 300 tataaagcgc cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac 360 tgccacaaac tgcatgagca aatggcagac tacaacgcgc tggggatcac cgtgcgttat 420 cttgctttcc cgcgccaggg gctggacagc gatgcagaga aagaaatgaa agctatctgg 480 tgtgcgaaag ataaaaacaa agcgtttgat gatgtgatgg caggtaaaag cgtcgcacca 540 gccagttgcg acgtggatat tgccgaccat tacgcacttg gcgtccagct tggcgttagc 600 ggtactccgg cagttgtgct gagcaatggc acacttgttc cgggttacca gccgccgaaa 660 gagatgaaag aattcctcga cgaacaccaa aaaatgacca gcggtaaata a 711 <210> 6 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> PelB <400> 6 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggcc 66 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgataacata tgaatagtga ctctgaatgt ccgg 34 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgataagtcg actcagcgtt cccaccactt caggtcacgg tactgacaac gctc 54 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tgataagtcg actcagcgca gttcccacca 30 <210> 10 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tgataacata tggatcctga caatgaggct tatgaaatgc cttctgagga agggtatcaa 60 gactacgaac ctgaagccaa tagtgactct 90 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgataacata tgaaaataaa aacaggtgca 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgataagaat tcagtctgcg cgtctttcag 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgataacata tgaaaaagat ttggctggcg 30 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tgataagaat tctccttttt tctcgcttaa gta 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tgataacata tgaagaaagg ttttatgttg ttt 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tgataagaat tctttccagc tggtcatttt ttg 33 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tgataaccat ggggaatagt gactctgaat gtccgg 36
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| KR101574204B1 (en) | 2013-12-02 | 2015-12-04 | 서울대학교 산학협력단 | Transgenic aves expressing codon-optimized human epidermal growth factor and method for preparing the same |
| WO2018084650A1 (en) * | 2016-11-04 | 2018-05-11 | 한국과학기술연구원 | Vector for expressing human epidermal growth factor or skin-permeable human epidermal growth factor, and microalgae transformed with vector |
| KR20190122420A (en) | 2018-04-20 | 2019-10-30 | 티앤에이치바이오(주) | Manufacture method of epidermal growth factor |
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| KR102049590B1 (en) * | 2017-12-29 | 2020-01-07 | (주)비씨알엠 | Production and purification process of high purity recombinant human epidermal growth factor |
| KR102099342B1 (en) * | 2019-09-03 | 2020-04-10 | 주식회사 제노포커스 | Expression Method of CRM197 Protein |
| CN117659212A (en) * | 2023-12-11 | 2024-03-08 | 吉林省中科康的科技有限公司 | Fusion protein of epidermal cell growth factor and preparation method and application thereof |
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| Protein Expression and purification, 1999, vol8, pp57-67. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101574204B1 (en) | 2013-12-02 | 2015-12-04 | 서울대학교 산학협력단 | Transgenic aves expressing codon-optimized human epidermal growth factor and method for preparing the same |
| WO2018084650A1 (en) * | 2016-11-04 | 2018-05-11 | 한국과학기술연구원 | Vector for expressing human epidermal growth factor or skin-permeable human epidermal growth factor, and microalgae transformed with vector |
| KR20190122420A (en) | 2018-04-20 | 2019-10-30 | 티앤에이치바이오(주) | Manufacture method of epidermal growth factor |
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