KR19990016368A - Method for producing human growth hormone using fusion protein - Google Patents
Method for producing human growth hormone using fusion protein Download PDFInfo
- Publication number
- KR19990016368A KR19990016368A KR1019970038898A KR19970038898A KR19990016368A KR 19990016368 A KR19990016368 A KR 19990016368A KR 1019970038898 A KR1019970038898 A KR 1019970038898A KR 19970038898 A KR19970038898 A KR 19970038898A KR 19990016368 A KR19990016368 A KR 19990016368A
- Authority
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- South Korea
- Prior art keywords
- expression vector
- growth hormone
- necrosis factor
- tumor necrosis
- human growth
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- 229910021654 trace metal Inorganic materials 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
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Abstract
본 발명은 종양괴사인자를 융합파트너로 하여 대장균에서 인성장호르몬 (human growth hormone, hGH)을 고수율로 생산하는 제조방법에 관한 것이다.The present invention relates to a method for producing human growth hormone (hGH) in E. coli with a high yield by using a tumor necrosis factor as a fusion partner.
보다 상세하게는, 본 발명은 종양괴사인자의 일부분과 인성장호르몬을 포함하는 융합단백질 유전자를 포함하는 발현 벡터, 그 발현 벡터로 형질전환된 미생물 그리고 발현된 융합단백질로부터 인성장호르몬을 정제하는 방법에 관한 것이다.More particularly, the present invention relates to an expression vector comprising a fusion protein gene comprising a part of a tumor necrosis factor and a human growth hormone, a microorganism transformed with the expression vector, and a method for purifying a human growth hormone from the expressed fusion protein .
본 발명의 발현 벡터는 종양괴사인자-α의 N-말단의 8번째에서 12번째에 해당하는 아미노산으로 구성된 펩타이드, 그의 C-말단에 10개의 히스티딘으로 된 폴리히스티딘 서열, 엔테로키나제 효소에 의해 특이적으로 절단되는 아스파라진산-아스파라진산-아스파라진산-아스파라진산-라이신(Asp-Asp-Asp-Asp-Lys) 서열 및 인성장호르몬 등을 코우딩하는 유전자를 포함한다.The expression vector of the present invention comprises a peptide consisting of the amino acids corresponding to the eighth to twelfth of the N-terminal of tumor necrosis factor-a, a polyhistidine sequence consisting of ten histines at its C-terminus, (Asp-Asp-Asp-Asp-Lys) sequence and a phosphorus-containing hormone, which are cleaved into asparaginic acid-aspartic acid-asparaginic acid-asparaginic acid-lysine.
본 발명의 제조방법에 의하면, N-말단에 메치오닌이 없는 천연형 인성장호르몬을 고수율로 용이하게 생산할 수 있다.According to the production method of the present invention, natural type growth hormone without methionine at the N-terminus can be easily produced at a high yield.
Description
본 발명은 종양괴사인자를 융합파트너로 하여 인성장호르몬 (human growth hormone, hGH)을 고수율로 제조하는 방법에 관한 것이다.The present invention relates to a method for producing a human growth hormone (hGH) with high yield by using a tumor necrosis factor as a fusion partner.
보다 상세하게는, 본 발명은 종양괴사인자의 일부분과 인성장호르몬의 융합 단백질 유전자를 포함하는 발현 벡터, 그 발현 벡터로 형질전환된 미생물 그리고 이로부터 발현된 융합단백질로부터 인성장호르몬을 정제하는 방법에 관한 것으로, 상기 발현 벡터는 폴리히스티딘 서열 및 엔테로키나제 절단 부위 등을 코우딩하는 유전자를 포함하여 이를 이용하면 N-말단에 메치오닌이 없는 천연형 인성장호르몬을 고수율로 용이하게 생산할 수 있다.More particularly, the present invention relates to a method for purifying a human growth hormone from an expression vector comprising a part of a tumor necrosis factor and a fusion protein gene of a human growth hormone, a microorganism transformed with the expression vector, and a fusion protein expressed therefrom Wherein the expression vector includes a gene coding for a polyhistidine sequence and an enterokinase cleavage site and the like, so that a natural type growth hormone without methionine at the N-terminus can be easily produced at a high yield.
인성장호르몬(human growth hormone)은 사람의 뇌하수체에서 생성되는 191개의 아미노산으로 구성되고 아미노 말단이 페닐알라닌-프롤린-트레오닌 (Phe-Pro-Thr)의 서열로 이루어져 있는 분자량 22,125 의 폴리펩티드 호르몬으로서, 단백질 합성, 세포증식 및 대사에 작용하여 인체의 정상적인 성장 및 유즙 분비 등에 중추적인 역할을 담당한다. hGH 는 이의 결핍으로 인한 어린이 왜소증 치료에 널리 사용되는 한편, hGH 결핍 성인 환자에게도 대체 투여하는 것으로 알려져 있다 (Raben, M. S. (1958) J. Clin. Endocr., 18, 901).Human growth hormone is a polypeptide hormone with a molecular weight of 22,125, consisting of 191 amino acids produced in the human pituitary gland and consisting of the amino-terminal sequence of phenylalanine-proline-threonine (Phe-Pro-Thr) , Acts on cell proliferation and metabolism, and plays a pivotal role in normal growth of human body and secretion of milk. hGH is widely used in the treatment of childhood obesity due to its deficiency, while it is also known to be an alternative to hGH deficient patients (Raben, M. S. (1958) J. Clin. Endocr., 18, 901).
인성장호르몬은 종래에 죽은 사람의 뇌하수체에서 추출 및 정제하여 사용되어 왔으나 생산되는 양이 극히 제한되어 모든 환자에게 공급되는데 어려움이 있었다. 또한 뇌하수체에서 분리한 인성장호르몬을 접종받은 어린이 4명이 바이러스에 감염되어 사망했다고 밝혀져, 미국 식품 의약국에서 상기의 방법으로 생산된 인성장호르몬의 사용을 금지시켰다 (Science, 234, 22, 1986). 따라서 최근에는 유전자 재조합 방법으로 대장균 및 효모 등에서 인성장호르몬을 발현시켜 정제하려는 많은 시도가 이루어졌으며, 이러한 인성장호르몬이 실제로도 임상에서 이용되고 있다 (대장균의 경우, 대한민국 특허공고 제 89-1244 호 및 제 87-701 호, 특허공개 제 87-2258 호 및 제 84-8695 호 참조; 효모의 경우, 대한민국 특허공고 제 92-99 호, 특허공개 제 90-9973 호 및 제 90-9976 호 참조).Human growth hormone has been used in extraction and purification from pituitary glands of dead people in the past, but the amount produced is extremely limited and has been difficult to supply to all patients. In addition, the US Food and Drug Administration banned the use of the human growth hormone produced by the above method (Science, 234, 22, 1986) because it was found that four children who had received a human pituitary gland hormone- . In recent years, many attempts have been made to express and purify human growth hormone in E. coli and yeast by recombinant methods, and such human growth hormone is actually used in clinical practice (in the case of E. coli, Korean Patent Publication Nos. 89-1244 and 87-701, 87-2258 and 84-8695; in the case of yeast, Korean Patent Publication Nos. 92-99, 90-9973 and 90-9976).
유전자 재조합 방법으로 사람 또는 동물장기에서 어렵게 얻어지던 여러 중요한 단백질들을 미생물에서 생산하려면 목적 단백질의 유전자를 발현 벡터에 클로닝하여 재조합 발현 벡터를 얻고 이를 적당한 숙주세포에 형질전환시켜 배양함으로써 이를 생산한다. 그러나 목적 단백질을 대장균에서 직접 발현시키는 경우, 생산되는 단백질이 숙주세포에 존재하는 단백질 분해효소에 의해 분해되는 현상이 나타나거나 (Gottesmann, S. (1989) Annu. Rev. Genet. 23, 163-198), N-말단에 메치오닌이 첨가된 형태로 만들어져 이를 천연형 단백질로 전환시키는 단계가 필요하다.In order to produce various important proteins that are difficult to obtain in human or animal organs by recombinant methods, the recombinant expression vector is obtained by cloning the gene of the desired protein into an expression vector and transformed into a suitable host cell and cultured. However, when the target protein is directly expressed in E. coli, the produced protein is degraded by the proteolytic enzyme present in the host cell (Gottesmann, S. (1989) Annu. Rev. Genet. 23, 163-198 ), Methionine is added to the N-terminal, and it is necessary to convert the protein into a native protein.
구체적으로 숙주세포로 대장균을 이용하는 경우, 만들고자 하는 단백질이 대장균내의 단백질 분해효소에 의해 분해되어 수율이 낮아지는 경우가 많으며, 특히 분자량 10,000 이하의 작은 크기의 폴리펩타이드의 발현에서 이러한 경향이 심한 것으로 알려져 있다 (Wetzel, R. Goeddel, D. V., Peptides 5, 1-64, 1983; Gottesmann, S. (1989) Annu. Rev. Genet. 23, 163-198). 이러한 현상을 줄이기 위해 목적 단백질을 세포 내에 불용성 응집체 (inclusion bodies) 형태로 발현하는 방법이 사용되는데, 이 때 단백질을 활성형으로 만들기 위해 구아니딘-하이드로클로라이드 (guanidine hydrochloride) 또는 우레아 (urea)와 같은 변성제 (denaturant) 등을 사용하여 희석 등을 통한 리폴딩 (refolding) 과정을 거치게 된다. 불용성 응집체로의 생산은 총불용성 단백질내에서 목적 단백질이 차지하는 비중이 상대적으로 높아 정제 목적으로 이용되기도 한다 (Pilanciski, W. P. et al. (1984) Bio/Technology 2, 356-360; Cabradila, C. D. et al. (1986) Bio/Technology 4, 128-133). 그러나 이후 거쳐야하는 리폴딩 과정은 아직까지 많은 문제점이 있어 생산 수율을 감소시키는 원인이 되기도 한다 (Marston, F. A. (1986) Biochem. J. 240, 1-12; Mitraki, A. and King, J. (1989) Bio/Technology 7, 690-697).Specifically, 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 such a tendency is severe in the expression of a small-sized polypeptide having a molecular weight of 10,000 or less (Wetzel, R. Goeddel, DV, Peptides 5, 1-64, 1983; Gottesmann, S. (1989) Annu. Rev. Genet., 23, 163-198). In order to reduce this phenomenon, a method of expressing a target protein in the form of insoluble inclusion bodies is used. In this case, a denaturant such as guanidine hydrochloride or urea is added to make the protein active. a refolding process such as dilution is performed using a denaturant or the like. Production of insoluble aggregates may be used for purification purposes due to the relatively high specific gravity of the target protein in the total insoluble protein (Pilanciski, WP et al. (1984) Bio / Technology 2, 356-360; Cabradila, CD et al (1986) Bio / Technology 4, 128-133). However, the subsequent refolding process still causes many problems, leading to a decrease in production yield (Marston, FA (1986) Biochem., 240, 1-12; Mitraki, A. and King, 1989) Bio / Technology 7, 690-697).
또한, 대장균내에서 발현되는 목적 단백질의 분해를 막고 안정성을 높여 수율을 향상시키기 위한 연구가 진행되어 목적 단백질을 대장균에서 융합단백질의 형태로 발현시키는 방법이 보고되었다 (Ulman, A. (1984) Gene 29, 27-31; Nilsson, B. et al. (1985) EMBO J. 4, 1075-1080; Di Guan et al. (1988) Gene 67, 21-30; La Vallie, E. R. et al. (1993) Bio/Technology 11, 187-193).Further, studies have been made to improve the yield by increasing the stability of the target protein expressed in E. coli, thereby expressing the target protein in the form of a fusion protein in E. coli (Ulman, A. (1984) Gene La Vallie, ER et al. (1993), < RTI ID = 0.0 > Bio / Technology 11, 187-193).
융합단백질은 보통 선택된 숙주에서 풍부하게 생산되는 것으로 알려진 단백질에 만들고자 하는 폴리펩타이드를 연결시킨 형태로 생산하는데, 지금까지 숙주에서 풍부하게 생성되는 것으로 알려진 단백질로는 베타-갈락토시다제 (β-galactosidase), 단백질 A (protein A), 글루타치온 에스-전달효소 (glutathione S-transferase) 등이 있다. 그러나 상기한 단백질을 융합파트너로 이용하는 경우 이들 융합단백질은 친화 크로마토그라피 등으로 용이하게 정제될 수 있는 장점이 있는 반면, 융합파트너가 제조하고자 하는 폴리펩타이드에 비해 상대적으로 크기가 커서 융합 부분의 크기에 따라 펩타이드의 수율이 현저히 떨어진다는 단점이 있다.Fusion proteins are usually produced in the form of a linkage of polypeptides that are intended to be produced in a protein known to be abundantly produced in a selected host. Examples of proteins known to be abundantly produced in host cells include beta-galactosidase ), Protein A (protein A), and glutathione S-transferase. However, when the above-mentioned protein is used as a fusion partner, these fusion proteins have an advantage that they can be easily purified by affinity chromatography or the like. On the other hand, the fusion partner has a relatively larger size than the polypeptide to be produced, There is a disadvantage in that the yield of the peptide is remarkably decreased.
한편 인성장호르몬은 191개의 아미노산으로 이루어진 당화가 되어있지 않은 단백질로서, 진핵세포에서 번역된 다음 변형과정 없이도 활성을 나타낼 수 있어 대장균과 같은 원핵생물을 이용하여 대량 생산할 수 있고 실제로 메치오닐-hGH 가 대장균에서 생산됨이 보고되었다 (Goeddel, D. V. et al. (1979) Nature 281, 544-548). 그러나 목적 단백질을 대장균에서 리더 펩타이드 없이 직접 발현시키는 경우 N-말단에 메치오닌이 첨가되어 인간이나 기타 동물에 적용되는 경우 면역 반응 (immunogenic)이 유발되거나 불안정하여 (unstable form) 본래의 단백질 기능을 수행하지 못하는 경우가 발생할 수 있다. 따라서 재조합 단백질을 천연형 단백질과 동일한 형태로 제조하는 방법을 개발하는 것은 매우 중요하다.On the other hand, human growth hormone is a non-glycosylated protein consisting of 191 amino acids. It can be produced in eukaryotic cells and can be produced in large quantities using prokaryotes such as E. coli. Methionyl-hGH Produced in E. coli (Goeddel, DV et al. (1979) Nature 281, 544-548). However, when the target protein is directly expressed in Escherichia coli without the leader peptide, when methionine is added to the N-terminus and applied to humans or other animals, immunogenicity is induced or unstable to perform the original protein function It can happen. Therefore, it is very important to develop a method for producing a recombinant protein in the same form as a native protein.
N-말단에 메치오닌이 제거된 천연형 인성장호르몬을 제조하기 위해 유전공학을 이용한 여러 방법들이 보고되었다 (Hsiung, H. M. et al. (1986) Bio/Technology 4, 991-995; Chang, C. N. et al. (1987) Gene 53, 189-196; Kato, C. et al. (1987) Gene 54, 197-202). N-말단의 메치오닌이 제거된 천연형 인성장호르몬을 제조하는 방법은 크게 인성장호르몬을 세포밖 또는 페리플라즘 (periplasmic space)으로 분비시키는 방법 (Hsiung, H. M. et al. (1986) Bio/Technology 4, 991-995; Nakayama, A. et al. (1987) J. Biotechnol. 5, 171-179; Gray, G. L. et al. (1984) Bio/Technology 2, 161-165) 또는 융합단백질의 형태로 세포내에 떨어뜨린후 리폴딩 과정 및 효소 처리 등으로 천연형 인성장호르몬을 생산하는 방법 (Franchi, E. et al. (1991) J. Biotechnol. 18, 41-54) 등으로 나눌 수 있다. 그러나 이들 방법은 분비되는 양이 적거나, 발현율이 낮은 단점이 있다.(1986) Bio / Technology 4, 991-995; Chang, CN et al. (1986)). In this study, (1987) Gene 53, 189-196; Kato, C. et al. (1987) Gene 54, 197-202). The method of producing N-terminal methionine-free natural type growth hormone is largely classified into a method of secreting human growth hormone into the extracellular space or the periplasmic space (Hsiung, HM et al. (1986) Bio / Technology Biotechnol. 5, 171-179; Gray, GL et al. (1984) Bio / Technology 2, 161-165) or in the form of fusion proteins (Franchi, E. et al. (1991) J. Biotechnol. 18, 41-54) can be categorized as a method of producing a natural type growth hormone by dropping in a cell and then refolding and enzymatic treatment. However, these methods have a disadvantage in that they are less secreted or have low expression rates.
종양괴사인자 (tumor necrosis factor, TNF)는 대식세포 등에서 분비되는 사이토카인의 일종으로서 항암작용 (Aggarwal, B. B. (1987) Drugs of the Future 891-898) 등을 포함한 여러 생물학적 활성을 지니고 있다. 종양괴사인자는 157개의 아미노산으로 구성되어 있는 베타-쉬이트 구조를 갖는 단백질로서 대장균내에서 높은 발현율을 가지고 안정적으로 생산되는 것으로 알려져 있다 (Pennica, D. et al. (1984) Nature 312, 724-729; Pennica, D. et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6060-6064).Tumor necrosis factor (TNF) is a kind of cytokine secreted by macrophages and the like and has various biological activities including anticancer action (Aggarwal, B. B. (1987) Drugs of the Future 891-898). Tumor necrosis factor is a protein having a beta-sheet structure composed of 157 amino acids and is known to be stably produced with a high expression rate in E. coli (Pennica, D. et al. (1984) Nature 312, 724- 729; Pennica, D. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6060-6064).
본 발명자들은 종양괴사인자의 전체 또는 일부 서열을 융합파트너로 이용하면 융합 부분의 크기를 기존방법에 비해 현저히 감소시킴으로 수율이 증대되고, 융합단백질이 가용성 형태로 발현됨으로 정제 공정이 단순화되는 것을 발견하여, 종양괴사인자를 융합파트너로 하여 작은 펩타이드들을 융합단백질 형태로 고수율로 발현시키는 방법에 대하여 특허출원한 바 있다 (특허출원 제 94-7018 호). 또한 상기 방법을 이용하여 글루카곤을 제조하는 방법에 대하여 이미 특허출원 및 논문으로 발표한 바 있다 (특허출원 제 95-40452 호; Kim, D. Y. et al. (1996) Biotechnol. Tech. 10, 669-672). 이외에 종양괴사인자와 아미노산 서열이 비슷하고 베타-쉬이트 구조를 갖는 림포톡신도 대장균내에서 안정적으로 발현되는 것으로 알려져 있어 상기 목적에 이용될 수 있다.The present inventors have found that when the whole or a part of the tumor necrosis factor sequence is used as a fusion partner, the yield is increased by significantly reducing the size of the fusion region compared to the conventional method, and the purification process is simplified because the fusion protein is expressed in a soluble form , And a method of expressing small peptides in a fusion protein form at a high yield using a tumor necrosis factor as a fusion partner (Patent Application No. 94-7018). (Patent Application No. 95-40452; Kim, DY et al. (1996) Biotechnol. Tech. 10, 669-672 (1996)) have already disclosed a method for producing glucagon using the above- ). In addition, it is known that lymphotoxin having a similar amino acid sequence to tumor necrosis factor and having a beta-sheet structure is stably expressed in E. coli, which can be used for the above purpose.
이에 본 발명자들은 인성장호르몬을 대량으로 생산하기 위하여, 종양괴사인자를 융합파트너로 이용하고 폴리히스티딘 서열 및 엔테로키나제 절단 부위를 인 성장호르몬 유전자의 5'-말단에 삽입한 발현 벡터를 제조하여 이를 대장균에 형질전환시키고 이로부터 융합단백질 형태의 인성장호르몬을 생산하여 정제한 다음 상기 N-말단 펩타이드를 엔테로키나제로 절단하여 공정이 단순하고 수율이 극대화된 천연형 인성장호르몬의 제조방법을 개발함으로써 본 발명을 완성하였다.Therefore, in order to produce a large amount of human growth hormone, the present inventors prepared an expression vector in which a tumor necrosis factor is used as a fusion partner and a polyhistidine sequence and an enterokinase cleavage site are inserted at the 5'-end of the growth hormone gene, Transformed into E. coli, producing a fusion protein-type human growth hormone, purified, and then cleaving the N-terminal peptide with an enterokinase to develop a process for producing a natural type growth hormone having a simple process and a maximized yield Thus completing the present invention.
본 발명은 종양괴사인자를 융합파트너로 이용하여 인성장호르몬을 고수율로 제조하는 방법을 제공함을 목적으로 한다.It is an object of the present invention to provide a method for producing a human growth hormone using a tumor necrosis factor as a fusion partner at a high yield.
도 1 및 도 2 는 발현 벡터 pT2GH 및 pT7T2GH 의 제조 과정을 나타낸 것이고,1 and 2 will showing the manufacturing process of an expression vector pT 7 and pT2GH T2GH,
도 3 은 본 발명에서 생산한 단백질 T2GH 의 시간에 따른 총발현율 및 가용성 부분의 비율을 나타낸 것이고,3 Shows the total expression ratio and the proportion of the soluble portion of the protein T2GH produced in the present invention over time,
도 4 는 상기 T2GH 의 DEAE-스트림라인 칼럼 크로마토그라피를 나타낸 것이고,4 Shows the DEAE-stream line column chromatography of the T2GH,
도 5 는 엔테로키나제 절단으로 얻은 hGH 의 Q-세파로스 FF 음이온 교환 크로마토그라피를 나타낸 것이고,Figure 5 shows the Q-Sepharose FF anion exchange chromatography of hGH obtained by enterokinase cleavage,
도 6 은 각 정제 단계에서의 T2GH 와 hGH 의 순도를 SDS-PAGE 로 분석한 결과를 나타낸 것이고,6 Shows the results of SDS-PAGE analysis of the purity of T2GH and hGH in each purification step,
레인 1 : 분자량 크기 마커; 레인 2 : 전체 대장균 파쇄액;Lane 1: molecular weight size marker; Lane 2: whole coliform lysate;
레인 3 : 수용성 단백질; 레인 4 : 불용성 침전;Lane 3: water soluble protein; Lane 4: insoluble sediment;
레인 5 : 금속 친화 칼럼으로 정제된 T2GH;Lane 5: T2GH purified with a metal-affinity column;
레인 6 : 엔테로키나제를 처리한 단백질 산물;Lane 6: protein product treated with enterokinase;
레인 7 : Q-세파로스 음이온 교환 크로마토그라피를 거친 hGHLane 7: hGH through Q-Sepharose anion exchange chromatography
도 7 은 정제된 hGH 의 HPLC 를 나타낸 것이고,Figure 7 shows the HPLC of purified hGH,
도 8 은 표준 hGH 및 정제된 hGH 의 트립신 분석을 나타낸 것이고,Figure 8 shows trypsin analysis of standard hGH and purified hGH,
도 9 는 정제된 hGH 의 수용체 결합능을 나타낸 것이고,Figure 9 shows the ability of the purified hGH to bind to the receptor,
도 10 은 정제된 hGH 의 원편광 이색분광 분석도를 나타낸 것이고,Figure 10 shows a circularly polarized dichromatographic analysis of purified hGH,
도 11 은 상기 hGH 의 정제 과정을 도식화하여 나타낸 것이다.FIG. 11 is a diagram illustrating the purification process of the hGH.
상기 목적을 달성하기 위하여 본 발명은 종양괴사인자 전체 또는 그의 일부가 치환된 형태의 종양괴사인자 뮤테인을 이용하여 인성장호르몬을 대장균에서 고수율로 제조하는 방법을 제공한다. 구체적으로 본 발명은 인간 종양괴사인자-α의 8번째에서 12번째에 해당하는 아미노산으로 구성되고 그의 C-말단에 폴리히스티딘 서열을 포함하는 펩타이드를 융합 파트너로 이용하여 엔테로키나제 절단 부위 DDDDK (Asp-Asp-Asp-Asp-Lys, 이하 D4K)가 있는 인성장호르몬을 생산하고 이로부터 인성장호르몬만을 용이하게 정제한다.In order to achieve the above object, the present invention provides a method for producing a human growth hormone in E. coli at a high yield by using a tumor necrosis factor mutein in which tumor necrosis factor or a part thereof is substituted. Specifically, the present invention relates to the use of a peptide containing a polyhistidine sequence at the C-terminal thereof as a fusion partner and having an enterokinase cleavage site DDDDK (Asp- Asp-Asp-Asp-Lys, hereinafter referred to as D4K), and only the human growth hormone is readily purified therefrom.
이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명은 분자량이 비교적 작고 대장균 내에서 가용성 단백질로 발현되는 종양괴사인자를 이용하여 인성장호르몬을 융합단백질 형태로 생산한다.The present invention produces a fusion protein in the form of a fusion protein using a tumor necrosis factor having a relatively small molecular weight and being expressed as a soluble protein in E. coli.
종양괴사인자는 157개의 아미노산으로 구성된 베타-쉬이트 구조를 가지고 있는 단백질로서 대장균내에서 안정적으로 발현되므로 종양괴사인자의 전체 아미노산 서열 또는 일부 아미노산 서열은 융합파트너로 효과적으로 이용될 수 있다.Tumor necrosis factor is a protein with a beta-sheet structure composed of 157 amino acids and is stably expressed in E. coli. Thus, the entire amino acid sequence or some amino acid sequence of tumor necrosis factor can be effectively used as a fusion partner.
본 발명은 종양괴사인자의 아미노산 서열중 N-말단의 8번째에서 12번째에 해당하는 아미노산으로 구성된 펩타이드 및 그의 C-말단에 10개의 히스티딘으로 된 폴리히스티딘 서열을 첨가한 펩타이드 (T2 펩타이드)를 인성장호르몬의 융합파트너로 이용함으로써 융합파트너 부분을 현저히 줄여 전체적인 수율을 증가시킨다.The present invention relates to a peptide comprising an amino acid sequence corresponding to the 8th to 12th amino acids of the N-terminal amino acid sequence of a tumor necrosis factor and a peptide (T2 peptide) to which 10 histidine polyhistidine sequences are added at its C- It can be used as a fusion partner of the intestinal hormone, thereby significantly reducing the fusion partner portion and increasing the overall yield.
구체적으로 본 발명은 상기 T2 펩타이드를 코우딩하는 유전자를 중합효소 연쇄반응 (Polymerase Chain Reaction, PCR)으로 증폭한 다음, 준비된 DNA 를 벡터에 삽입하여 상기 융합파트너를 생산할 수 있는 발현 벡터 pT2 및 pT7T2 를 제조한다 (도 1 참조).More specifically, the present invention provides expression vectors pT2 and pT 7 to produce the fusion partner by inserting the amplified genes that Koh guiding the T2 peptide by PCR (Polymerase Chain Reaction, PCR) and then, vector prepared DNA T2 (see Fig. 1).
또한, 본 발명은 인성장호르몬 유전자의 5'-말단에 엔테로키나제 절단 부위인 D4K 를 코우딩하는 염기 서열을 첨가한 DNA 를 PCR 로 증폭한 다음 이 유전자를 상기 발현 벡터에 적당히 삽입하여, 인성장호르몬이 상기 D4K 서열을 포함하는 융합단백질로 발현된 후 엔테로키나제에 의한 절단으로 천연형 인성장호르몬을 생산할 수 있는 발현 벡터를 제조한다.The present invention also relates to a method of amplifying a DNA comprising the nucleotide sequence encoding D4K, which is an enterokinase cleavage site, at the 5'-end of the human growth hormone gene, by PCR, and then inserting the gene into the expression vector, After expressing the hormone with a fusion protein comprising the D4K sequence, an expression vector capable of producing a natural type growth hormone by cleavage with an enterokinase is prepared.
구체적으로, 본 발명은 상기 발현 벡터 pT2 또는 pT7T2 의 제한효소Bam HI및Hind Ⅲ자리에 상기 D4K 서열과 결합한 인성장호르몬 유전자를 삽입하여 발현 벡터 pT2GH 또는 pT7T2GH 를 얻는다 (도 2 참조).More specifically, the present invention by inserting the human growth hormone gene in combination with the D4K sequence with restriction enzymes Bam HI and Hind Ⅲ place in the expression vector pT2 or pT 7 T2 to obtain the expression vector pT2GH or pT 7 T2GH (see Fig. 2) .
이외에도 상기 T2 펩타이드와 인성장호르몬의 융합단백질 (T2GH 단백질) 유전자를 코우딩하는 DNA 를 lac, trp, tac, pL, T3, T7, SP6, SV40, λ (pL/pR) 등의 적당한 프로모터를 갖는 발현 벡터에 직접 삽입하거나, 이들 프로모터와 리보좀 결합부위를 갖는 DNA 를 융합단백질 DNA 와 결합시킨 다음 다양한 플라스미드에 삽입하여 발현 벡터 시스템을 구축할 수 있다.In addition, the DNA encoding the T2 peptide and the fusion protein (T2GH protein) gene of the human growth hormone may be modified to have a suitable promoter such as lac, trp, tac, pL, T3, T7, SP6, SV40, Or an expression vector system can be constructed by inserting DNA having these promoter and ribosome binding site into a fusion protein DNA and then inserting it into various plasmids.
상기에서 N-말단의 인간 종양괴사인자-α의 올리고펩타이드 (PSDKP)는 대장균에서 T2GH 단백질을 가용성 단백질로 발현을 유도하는 리더 펩타이드로 작용하고, 폴리히스티딘 서열(H10)은 금속 킬레이트 수지 (metal-chelated resin) 친화성 및 낮은 pH 에서 히스티딘이 양전하를 띄는 성질을 이용하여 양이온 교환수지 등을 이용하여 T2GH 단백질이 용이하게 정제되도록 한다. 또한, 엔테로키나제 절단 부위인 D4K 는 T2GH 단백질이 엔테로키나제에 의해 절단되어 천연형 인성장호르몬을 생성하는데 유용하다.In this case, the N-terminal oligopeptide of human tumor necrosis factor-a (PSDKP) acts as a leader peptide for inducing the expression of T2GH protein as a soluble protein in E. coli, and the polyhistidine sequence (H10) chelated resin) It is easy to purify the T2GH protein by using cation exchange resin or the like using affinity and affinity of histidine at low pH. In addition, D4K, an enterokinase cleavage site, is useful for cleaving T2GH protein by enterokinase to produce a natural type growth hormone.
상기 인성장호르몬 융합단백질을 생산하는 발현 벡터를 적당한 숙주에 하나한 (Hanahan, D. (1985) DNA Cloning vol. 1, 109-135, IRS press)의 방법으로 도입하여 형질전환체를 제조하였다.Transformants were prepared by introducing the expression vector for producing the human FSH fusion protein into an appropriate host (Hanahan, D. (1985) DNA Cloning vol. 1, 109-135, IRS press).
구체적으로 상기 발현 벡터 pT2GH 를 대장균 BL21(DE3) 균주에 형질전환시켜 얻은 대장균 형질전환체를 한국과학기술연구원 부설 생명공학연구소 유전자은행에 1997년 2월 25일에 기탁하였다 (수탁번호 : KCTC 8786 P).Specifically, an E. coli transformant obtained by transforming the above expression vector pT2GH into E. coli BL21 (DE3) strain was deposited on Feb. 25, 1997 under the accession number of Genetic Bank, Institute of Bioscience and Biotechnology, Korea Research Institute of Science and Technology (accession number: KCTC 8786 P ).
상기 대장균 형질전환체를 적당한 배양조건에서 배양하여 인성장호르몬을 포함하는 융합단백질을 생산할 수 있다. 배양된 세포들은 라이소자임 소화, 동결융해, 초음파 파쇄, 또는 프렌치 프레스 등으로 처리한 후, 원심분리나 여과를 통해 상기 융합단백질을 함유하는 추출물을 얻는다. 상기 융합단백질은 추출물을 용해, 초여과, 투석, 이온 교환 크로마토그라피, 겔 여과, 전기영동 그리고 친화성 크로마토그라피 등의 일반적인 정제방법을 통해 분리해 낼 수 있다. 분리된 융합단백질을 적당량의 엔테로키나제로 처리하면 이로부터 인성장호르몬만을 얻을 수 있다. 정제된 인성장호르몬은 브래드포드 단백질 정량방법 (Bradford, M. (1976) Anal. Biochem. 72, 248-254) 등에 의해 정량할 수 있으며, 아미노산 분석, N-말단 아미노산 서열 분석, 질량 분석, 펩타이드 맵핑, 수용체 결합 분석 등을 통해 천연형 인성장호르몬임을 확인한다.The E. coli transformant may be cultured under appropriate culture conditions to produce a fusion protein comprising the phosphorous endocrine hormone. The cultured cells are treated with lysozyme digestion, freeze thawing, ultrasonic disruption, or French press, and then an extract containing the fusion protein is obtained by centrifugation or filtration. The fusion protein can be isolated by a general purification method such as dissolution, ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis and affinity chromatography. When the isolated fusion protein is treated with an appropriate amount of enterokinase, only the human growth hormone can be obtained therefrom. Purified human growth hormone can be quantitated by Bradford, M. (1976) Anal. Biochem. 72, 248-254) and can be analyzed by amino acid analysis, N-terminal amino acid sequence analysis, mass spectrometry, Mapping, receptor binding analysis, etc., to confirm that it is a natural type growth hormone.
본 발명의 T2GH 단백질은 대장균에서 가용성으로 발현된 후 세포내에서 서서히 침전되는 특징이 있어 원심분리 등의 방법으로 손쉽게 분리해 낸 다음 알칼리성 용액에 녹여 위와 같은 방법으로 정제될 수 있다.The T2GH protein of the present invention is characterized in that it is solubilized in E. coli and slowly precipitated in the cell. Therefore, the T2GH protein can be easily separated by centrifugation or the like, dissolved in an alkaline solution, and purified by the above method.
이 때 T2GH 가 초기에 가용성으로 발현된 후 세포내에서 불용성으로 침전하는 현상은 본 발명의 특이적인 것으로서 단백질 발현시 나타나는 불용성 응집체 (inclusion bodies)와는 달리 알칼리성 용액에 쉽게 용해되므로 유용하다.At this time, the phenomenon that T2GH is initially expressed solubilized and then precipitated insoluble in the cells is specific to the present invention and is useful because it is easily dissolved in an alkaline solution, unlike insoluble inclusion bodies appearing in protein expression.
이하 본 발명을 실시예에 의거하여 좀 더 상세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail with reference to examples.
그러나 실시예는 본 발명을 예시하는 것일뿐, 본 발명이 실시예에 의해 한정되는 것은 아니다.However, it is to be understood that the present invention is not limited by these Examples.
실시예 1 종양괴사인자 유전자를 융합파트너로 이용한 발현 벡터의 제조Example 1 Preparation of expression vector using tumor necrosis factor gene as a fusion partner
인간 종양괴사인자-α (human tumor necrosis factor-alpha, 이하 hTNF-α라고 한다)를 주형으로 하여 N-말단 8번째에서 12번째에 해당하는 아미노산으로 구성된 펩타이드와 이 펩타이드의 C-말단에 10개의 히스티딘으로 된 폴리히스티딘 서열을 첨가한 T2 폴리펩타이드를 코우딩하는 유전자를 다음과 같은 프라이머를 이용하여 PCR 방법으로 증폭하였다.A peptide consisting of an amino acid corresponding to the 8th to 12th amino acids of the N-terminal and a human tumor necrosis factor-alpha (hereinafter referred to as hTNF-alpha), and a peptide consisting of 10 amino acids at the C- The gene coding for the T2 polypeptide added with the histidine-derived polyhistidine sequence was amplified by the PCR method using the following primers.
T2-I (센스 프라이머): 5'-TATACATATGCCGAGTGACAAGCCT-3'T2-I (sense primer): 5'-TATACATATGCCGAGTGACAAGCCT-3 '
T2-Ⅱ (센스프라이머): 5'-AGTGACAAGCCTCATCATCATCATCATCATCATCATCAT CATCACAGCAG-3'T2-II (sense primer): 5'-AGTGACAAGCCTCATCATCATCATCATCATCATCATCAT CATCACAGCAG-3 '
T2-Ⅲ (안티센스프라이머): 3'-TAGTAGTGTCGTCGCCTAGGTACA-5'T2-Ⅲ (antisense primer): 3'-TAGTAGTGTCGTCGCCTAGGTACA-5 '
이 때 프라이머 T2-I, T2-Ⅱ 그리고 T2-Ⅲ 를 10 : 1 : 10 의 비율로 섞어 중합효소 연쇄반응을 실시하였다. 증폭된 DNA 절편을 제한효소NdeI과BamHI으로 각각 처리하여 5′말단에NdeI제한효소 부위와 3′말단에BamHI제한 효소 부위를 갖도록 하여 벡터 pET-3a (Novagen, 미국)의 제한효소NdeI과BamHI자리에 삽입하였다. 이렇게 만들어진 플라스미드를 pT2 이라고 명명하였다 (도 1 참조).The primers T2-I, T2-Ⅱ and T2-Ⅲ were mixed at a ratio of 10: 1: 10. Respectively The amplified DNA fragment with restriction enzymes NdeI and BamHI 5 restriction enzymes NdeI and BamHI position of 'NdeI restriction site and 3 at the ends, to have a BamHI restriction site at the terminal vector pET-3a (Novagen, USA) Lt; / RTI > The plasmid thus constructed was named pT2 (see Fig. 1).
같은 방법으로 T2 폴리펩타이드를 코우딩하는 유전자를 제한효소NdeI과BamHI으로 각각 처리하여 5′말단에NdeI제한효소 부위와 3′말단에BamHI제한 효소 부위를 갖도록 한 다음 벡터 pT7-7 (Tabor, S. and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078, 하바드의과대학 생물화학과에서 입수가능함)의 제한효소NdeI과BamHI자리에 삽입하였다.In the same manner, the gene encoding the T2 polypeptide was treated with restriction enzymes NdeI and BamHI , respectively, and the NdeI restriction enzyme site and the BamHI restriction enzyme site were added at the 5 'end and the 3' end, respectively, and then the vector pT7-7 (Tabor, S USA, 82, 1074-1078, available from Harvard Medical School, Department of Biochemistry), and the restriction enzymes NdeI and BamHI .
이렇게 만들어진 발현 벡터를 pT7T2 이라고 명명하였다 (도 2 참조).The expression vector thus constructed was named pT 7 T2 (see FIG. 2).
실시예 2 인성장호르몬을 융합단백질 형태로 생산하는 발현 벡터의 제조Example 2 Preparation of Expression Vectors Producing Human Growth Hormone in Fusion Protein Form
인성장호르몬 유전자는 인간의 뇌하수체 (pituitary gland) cDNA 라이브러리(Clontech, 미국)를 주형으로 하고, 다음과 같은 프라이머를 이용하여 PCR 방법으로 제조하였다.The human growth hormone gene was prepared by PCR using the following primers with a human pituitary gland cDNA library (Clontech, USA) as a template.
GH-I (센스프라이머):GH-I (sense primer):
5' -GCAGGATCCGACGACGACGACAAATTCCCAACCATTTCCCTTATC- 3′5 '-GCAGGATCCGACGACGACGACAAATTCCCAACCATTTCCCTTATC-3'
D D D D K [→→→ hGHD D D D K [→→→ hGH
GH-Ⅱ (안티센스프라이머):GH-II (antisense primer):
5' -TGCAAGCTTTTACTAGAAGCCACAGCTGCCCTCCACAG- 3′5 '-TGCAAGCTTTTACTAGAAGCCACAGCTGCCCTCCACAG-3'
프라이머 GH-I 은 인성장호르몬 서열의 5′말단에 엔테로키나제 절단 부위인 D4K 를 코우딩하는 DNA 서열을 추가하여 인성장호르몬이 D4K 를 포함하는 융합단백질로 발현된 후 엔테로키나제 절단에 의해 메치오닌이 없는 천연형 인성장호르몬이 생성되도록 고안되었다.In the primer GH-I, a DNA sequence encoding D4K, an enterokinase cleavage site, was added to the 5 'end of the human growth hormone sequence, so that the human growth hormone was expressed as a fusion protein containing D4K and then cleaved by enterokinase cleavage It is designed to produce a naturally occurring form of human growth hormone.
증폭된 DNA 를 제한효소Bam HI및Hind Ⅲ로 절단한 뒤 상기 플라스미드 pT2 의 제한효소Bam HI과Hind Ⅲ자리에 삽입하였다. 이렇게 만들어진 발현 벡터를 pT2GH 라고 명명하였다 (도 2 참조).Cutting the amplified DNA with restriction enzymes Bam HI and Hind Ⅲ back was inserted into the restriction enzyme Bam HI and Hind Ⅲ place of the plasmid pT2. The expression vector thus constructed was named pT2GH (see Fig. 2).
같은 방법으로, 증폭된 DNA를 제한효소BamHI과HindⅢ로 절단한 뒤 상기 플라스미드 pT7T2 의Bam HI과Hind Ⅲ자리에 삽입하였다. 이렇게 만들어진 발현 벡터를 pT7T2GH 라고 명명하였다 (도 2 참조).The same way, was inserted into the amplified DNA with restriction enzymes BamHI and HindⅢ a plasmid behind the Bam HI and Hind Ⅲ place of pT 7 T2 cut into. The expression vector thus constructed was named pT 7 T2GH (see Fig. 2).
실시예 3 대장균 형질전환체의 제조Example 3 Preparation of Escherichia coli Transformant
하나한(Hanahan)이 기술한 방법에 의해 발현 벡터 pT2GH 로 대장균 BL21(DE3)을 형질전환 (Hanahan, D. (1985) DNA Cloning vol.1 109-135, IRS press)시킨 다음 앰피실린에 저항성이 있는 콜로니를 선별하였다.E. coli BL21 (DE3) was transformed with the expression vector pT2GH by the method described by Hanahan (Hanahan, D. (1985) DNA Cloning vol.19-135, IRS press) and then resistant to ampicillin Were selected.
상기 발현 벡터 pT2GH 로 형질전환된 대장균 BL21(DE3) 균주를 선택하여 1997년 2월 25일에 생명공학연구소 유전자은행에 기탁하였다 (수탁번호 KCTC 8786P).E. coli strain BL21 (DE3) transformed with the expression vector pT2GH was selected and deposited on February 25, 1997 with the gene bank of the Institute of Biotechnology (Accession No. KCTC 8786P).
실시예 4 대장균 형질전환체의 배양 및 T2GH 단백질의 생산Example 4 Culture of Escherichia coli Transformant and Production of T2GH Protein
가) 균주 및 배지A) Strain and medium
재조합 발현 벡터 pT2GH 로 형질전환된 대장균 형질전환체를 이용하여 유가식 배양으로 T2GH 를 발현시킴으로 대량 생산하였다. 종균 배지의 조성은 1L당 트립톤 10g, 효모 추출물 10g, NaCl 5g 의 LB 배지이며 회분 및 유가식 배양을 위해 이용된 배지의 조성은 아래와 같다.The recombinant expression vector pT2GH was transformed with E. coli transformants and mass produced by expressing T2GH by fed-batch culture. The composition of the seed culture medium is 10 g of tryptone per liter, 10 g of yeast extract and 5 g of NaCl, and the composition of the medium used for ash and fed-batch culture is as follows.
(1) 회분 배양 (batch cultivation)(1L 당)(1) batch cultivation (per liter)
① KH2PO413.3 g1 KH 2 PO 4 13.3 g
(NH4)2HPO44 g (NH 4) 2 HPO 4 4 g
시트르산 1.7 gCitric acid 1.7 g
티아민 HCl 0.1 gThiamine HCl 0.1 g
소량 금속 용액 20.0 mLSmall amount of metal solution 20.0 mL
② MgSO4.7H2O 1.2 g② MgSO 4 .7H 2 O 1.2 g
③ 포도당 20 g③ glucose 20 g
이 때 상기 성분 ①, ②, ③ 을 분리하여 각각 121℃ 에서 15분간 멸균한 후 혼합하였고, 배지를 혼합한 다음 200mg/L 농도가 되도록 필터 주사기(syringe filter , 0.2μm)로 앰피실린을 첨가하였다. 상기 소량 금속 용액 (trace metal solution , 50X)은 1L 중에 FeCl3.6H2O2.2 g, ZnSO4.7H2O 1.05 g, CuSO4.5H2O 0.18 g, Na2MoO4.2H2O 0.2 g, CoCl2.6H2O 0.2 g, MnCl2.4H2O1.17 g, H3BO30.25 g, EDTA 0.65 g 을 포함한다.At this time, the components (1), (2) and (3) were separated and sterilized at 121 ° C for 15 minutes and then mixed. After mixing the medium, ampicillin was added to a syringe filter (0.2 μm) . The trace metal solution (50X) contained, in 1 L, FeCl 3 .6H 2 O.2 g, ZnSO 4 .7H 2 O 1.05 g, CuSO 4 .5H 2 O 0.18 g, Na 2 MoO 4 .2H 2 O 0.2 g, CoCl 2 .6H include 2 O 0.2 g, MnCl 2 .4H 2 O1.17 g, H 3 BO 3 0.25 g, EDTA 0.65 g.
(2) 유가식 배양 (fed-batch cultivation)(2) Fed-batch cultivation
1) 세포 성장기 (growth phase) (1L 당)1) Cell growth phase (per 1L)
① 소량 금속 용액 20 mL① Small amount of metal solution 20 mL
MgSO4.7H2O 20 gMgSO 4 .7H 2 O 20 g
② 포도당 800g② glucose 800g
이 때 상기 성분 ①, ② 를 분리하여 각각 121℃에서 15분간 멸균한 후 혼합하였고, 배지를 혼합한 다음 1g/L 농도가 되도록 필터 주사기 (0.2μm)로 앰피실린을 첨가하였다. 또한, NH4OH 를 질소원 및 pH 조절자로 동시에 이용하였다.The components (1) and (2) were separated and sterilized at 121 ° C for 15 minutes and then mixed. The medium was mixed and then ampicillin was added to the filter syringe (0.2 μm) so as to have a concentration of 1 g / L. NH 4 OH was also used as a nitrogen source and pH adjuster at the same time.
2) 재조합 유전자의 발현기 (induction phase or product formation phase)에서 사용한 배지 (1L 당)2) In the induction phase or product formation phase of the recombinant gene,
① (NH4)2SO41.5 g(NH 4 ) 2 SO 4 1.5 g
이스트 추출물 다양한 양Various amounts of yeast extract
MgSO4.7H2O 1 gMgSO 4 .7H 2 O 1 g
② 포도당 다양한 양② Various amounts of glucose
상기 성분 ①, ②를 분리하여 각각 121℃ 에서 15분간 멸균한 후 섞어 혼합하였다. 배지를 혼합한 다음 100mg/L 의 농도가 되도록 필터 주사기 (0.2μm)로 앰피실린을 첨가하였다.The components (1) and (2) were separated, sterilized at 121 ° C for 15 minutes, and mixed. The medium was mixed and then ampicillin was added to the filter syringe (0.2 mu m) to a concentration of 100 mg / L.
나) 대장균 형질전환체의 배양 및 T2GH 발효B) Culture of Escherichia coli transformants and T2GH fermentation
재조합 대장균은 종균 배지와 동일한 성분의 아가 플레이트에서 배양한 다음 생성된 콜로니를 15% 글리세롤 용액에 현탁시켜 -70℃에 보관하였다. 배양시에는 -70℃에 보관중인 재조합 대장균을 상기와 동일한 아가 플레이트에 도말하여 37℃에서 밤새 배양한 다음 생성된 콜로니를 종균 배지에 다시 접종하여 37℃, 200 rpm으로 교반하면서 배양하였다. 16시간이 경과한 다음 이 종균 배지를 10% 농도가 되도록 회분 배양 배지가 들어있는 5 L 발효조에 접종하여 37℃, pH 6.75 조건에서 배양을 시작하였다. 배지 중의 포도당이 모두 소모되면 유가식 배양의 세포 성장기 배지를 발효조의 조절 장치(control module)에 연결된 펌프를 이용하여 주입하는데, 아래식에 따라 배양액 중의 포도당 농도를 100 mg/L 이하로 유지하며 주입속도가 세포성장에 따라 점차 지수적으로 증가하도록 조절하며 주입하였다. 동시에 암모니아수를 질소원 및 pH 조절자로 이용하였다.The recombinant E. coli was cultured in agar plates of the same composition as the seed culture medium, and the resulting colonies were suspended in a 15% glycerol solution and stored at -70 ° C. At the time of culturing, the recombinant E. coli stored at -70 ° C was plated on the same agar plate as above and cultured overnight at 37 ° C. The resulting colonies were inoculated again into the seed culture medium and cultured at 37 ° C. with stirring at 200 rpm. After 16 hours, the seed culture medium was inoculated into a 5 L fermenter containing a batch culture medium at a concentration of 10%, and cultivation was started at 37 ° C and pH 6.75. When the glucose in the medium is consumed, the cell growth medium of the fed-batch culture is injected using a pump connected to the control module of the fermenter. The glucose concentration in the culture medium is maintained at 100 mg / L or lower The rate was adjusted to increase exponentially with cell growth. At the same time, ammonia water was used as a nitrogen source and pH adjuster.
S0: 주입 배지 중의 포도당 농도 (g/L)S 0 : glucose concentration in the infusion medium (g / L)
X : 배지 중의 대장균 농도 (g/L)X: concentration of E. coli in the medium (g / L)
μ : 비성장속도 (hr-1)μ: non-growth rate (hr -1 )
V : 배지 부피 (L)V: Medium volume (L)
F : 배지 주입속도 (L/hr)F: Media feeding rate (L / hr)
Δt : 30초Δt: 30 seconds
세포 성장기 중 대장균이 70-80 g/L 의 농도까지 성장하였을 때 주입배지를 상기 단백질 생산기의 배지로 바꾸어 주면서 재조합 대장균 g 당 5mmol 양으로 IPTG 를 첨가하여 T7 프로모터의 발현을 유도하였다. 또한 재조합 대장균 g 당, 단위시간(h) 당 0.3mL 을 유지하도록 배지의 주입 속도를 조절하였다. 발효조에서의 배양은 모두 용존산소가 40% 이상이 되도록 교반속도를 조절하면서 수행하였고 2-4시간 간격으로 시료를 취하여 세포 농도, 아세트산 농도, T2GH 농도, 단백질 용해도 등을 분석하였다. 결과적으로 아세트산 축적을 억제하면서 발효배지 내에서 T2GH 를 약 9 g/L 까지 생산하였다. 초기에 T2GH 는 대장균 세포내에서 가용성으로 발현되었으나 점차 침전되어 배양 말기에는 80% 이상이 불용성 침전체의 형태로 존재하였다.When Escherichia coli was grown to a concentration of 70-80 g / L in the cell growth period, the expression medium of T7 promoter was induced by adding IPTG in an amount of 5 mmol per g of recombinant E. coli while changing the culture medium to the medium of the protein producer. In addition, the feeding rate of the medium was adjusted so as to maintain 0.3 mL per unit time (h) per g of recombinant E. coli. In the fermentation tank, the concentration of dissolved oxygen was over 40%, and the stirring rate was adjusted. Cells concentration, acetic acid concentration, T2GH concentration and protein solubility were analyzed at 2-4 hour intervals. As a result, T2GH was produced to about 9 g / L in the fermentation medium while inhibiting acetic acid accumulation. Initially, T2GH was expressed solubilized in E. coli cells, but gradually precipitated and more than 80% of the cells were present in the form of insoluble sediments at the end of the culture.
〈실시예 5〉 인성장호르몬의 정제≪ Example 5 > Purification of human growth hormone
1) 세포 파쇄 (cell disruption)1) Cell disruption
고농도 배양에서 얻는 세포 배양액을 6000 Xg 에서 20분간 원심분리하여 펠렛을 회수하였다. 회수된 대장균은 완충액 A (20mM 트리스-HCl, pH 8.0, 1mM PMSF, 1mM EDTA, 0.5% 트리톤 X-100)로 4배 희석하여 4℃에서 저어주어 세포 현탁액을 만들어 주었다. 세포 현탁액을 프렌치 프레스 (Niro-Soavi, Italy)를 이용하여 1000바에서 2회 파쇄하였다. 이후 각 단계들의 단백질 정량은 브래드포드 방법 (Bradford method)을 사용하였으며, 상기 단백질 파쇄액은 14% SDS-PAGE 를 수행하여 분석하였다 (도 6 참조).The cell culture obtained from the high-concentration culture was centrifuged at 6000 xg for 20 minutes to recover the pellet. The recovered Escherichia coli was diluted 4-fold with buffer A (20 mM Tris-HCl, pH 8.0, 1 mM PMSF, 1 mM EDTA, 0.5% Triton X-100) and stirred at 4 ° C to prepare a cell suspension. The cell suspension was ruptured twice at 1000 bar using a French press (Niro-Soavi, Italy). Protein quantification at each step was performed using the Bradford method, and the protein disruption solution was analyzed by performing 14% SDS-PAGE (see FIG. 6).
2) 불용성 침전체의 분리2) Isolation of insoluble sediment
상기 세포 파쇄액을 8000 Xg에서 20분간 원심분리하여 펠렛을 회수하고, 이 펠렛을 완충액 B (1mM EDTA 가 없는 완충액 A)로 4배 희석하여 현탁액을 만들고 다시 원심분리하여 펠렛을 취하는 방법을 2회 반복하였다. 이렇게 얻어진 펠렛을 완충액 C (0.5% 트리톤 X-100 이 없는 완충액 B)로 4배 희석하여 현탁액을 만들어 원심분리한 다음 펠렛을 취하였다 (도 3 참조).The cell lysate was centrifuged at 8000 xg for 20 minutes to recover the pellet, and the pellet was diluted 4-fold with buffer B (buffer A without 1 mM EDTA) to prepare a suspension. The pellet was recovered by centrifugation again twice And repeated. The pellet thus obtained was diluted 4-fold with buffer C (Buffer B without 0.5% Triton X-100) to prepare a suspension, followed by centrifugation and taking the pellet (see Fig. 3).
3) 용해3) Fusion
위에서 얻어진 침전체 펠렛을 20mM 트리스 완충액에 2g/L가 되게 현탁하고, NaOH 를 가하여 pH 를 12 로 만들어 침전체를 용해시켰다. 이 용액을 천천히 저어주면서 20mM 트리스-HCl, pH 8.0 을 가하여 4배 희석시켰다.The saliva total pellet obtained above was suspended in 20 mM Tris buffer to a concentration of 2 g / L and NaOH was added to adjust the pH to 12 to dissolve the needle. The solution was diluted 4 times with 20 mM Tris-HCl, pH 8.0, while slowly stirring.
4) 니켈-킬레이팅 스트림라인 친화 컬럼 크로마토그라피 (Nickel-chelating Streamline affinity chromatography)4) Nickel-chelating Streamline Affinity Chromatography (Nickel-chelating Streamline affinity chromatography)
상기 T2GH 용액을 정제하기 위하여 니켈-킬레이팅 스트림라인(Nickel-chelating Streamline)을 이용하였다. 먼저 A50 칼럼 (0.6L, Pharmacia, Sweden)에 칼럼 부피의 5배인 50mM NiCl2을 통과시켜 니켈을 결합시키고 (nickel charging), 결합 완충액 (20mM 트리스-HCl, 0.5M NaCl, pH 8.0)으로 칼럼을 평형화 시켰다. 평형화된 칼럼에 NaCl 을 0.5M 되게 가한 T2GH 용액을 로딩한 다음 세척 완충액(0.05M 이미다졸을 가한 결합 완충액)으로 칼럼을 세척하여 불순물을 제거하고 용출 완충액 (0.5M 이미다졸을 가한 결합 완충액)으로 T2GH 를 용출하였다 (도 4 참조).A nickel-chelating stream line was used to purify the T2GH solution. First, 50 mM NiCl 2, which is 5 times the column volume, was passed through an A50 column (0.6 L, Pharmacia, Sweden) to bind nickel (nickel charging) and column with binding buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) Lt; / RTI > The equilibrated column was loaded with a solution of T2GH added with 0.5 M NaCl, and then the column was washed with washing buffer (binding buffer containing 0.05 M imidazole) to remove impurities and eluted with elution buffer (binding buffer containing 0.5 M imidazole) T2GH was eluted (see Fig. 4).
5) 엔테로키나제 절단 (Enterokinase cleavage)5) Enterokinase cleavage
니켈-킬레이팅 스트림라인 (Nickel-chelating Streamline)을 통하여 정제된 T2GH 는 20mM 트리스-HCl, pH 8.0 에서 투석 (dialysis)을 수행하여 NaCl 을 제거한 후 정제된 엔테로키나제를 이용하여 T2GH 로부터 hGH 폴리펩타이드를 절단해내는 반응을 수행하였다. 반응은 0.4g/L 농도의 T2GH 에 1/500 (T2GH/엔테로키나제, w/w)의 엔테로키나제를 가하고 37℃에서 16-24시간 동안 수행시켰다.T2GH purified through Nickel-chelating Streamline was subjected to dialysis at 20 mM Tris-HCl, pH 8.0 to remove NaCl and then purified hGH polypeptide from T2GH using purified enterokinase The cleavage reaction was carried out. The reaction was performed by adding 1/500 (T2GH / enterokinase, w / w) of enterokinase to T2GH at a concentration of 0.4 g / L and incubating at 37 ° C for 16-24 hours.
6) Q-세파로스 FF 음이온 교환 크로마토그라피 (Q-Sepharose FF anion exchange chromatography)6) Q-Sepharose FF anion exchange chromatography (Q-Sepharose FF anion exchange chromatography)
엔테로키나제 절단에 의하여 생성된 hGH 를 순수하게 정제하기 위하여 음이온교환 크로마토그라피를 수행하였다. 먼저 엔테로키나제에 의한 반응이 끝난 반응액을 10mM 인산나트륨, pH 7.0 으로 4배 희석하고 같은 완충액으로 미리 평형화된 Q-세파로즈 FF 칼럼 (50/50, 250mL, Pharmacia, Sweden)에 로딩하고 세척을 수행한 후, 5-0.5M NaCl의 선형 농도구배를 형성시켜서 hGH 를 용출하였다. 용출된 hGH 분획들을 취합하여 10mM 글라이신에서 투석 (dialysis)한 후, 동결건조하였다 (도 5 참조).Anion exchange chromatography was performed to purely purify hGH produced by enterokinase cleavage. First, the reaction mixture was diluted four times with 10 mM sodium phosphate, pH 7.0, loaded onto a Q-Sepharose FF column (50/50, 250 mL, Pharmacia, Sweden) pre-equilibrated with the same buffer, After performing a linear concentration gradient of 5-0.5 M NaCl, hGH was eluted. The eluted hGH fractions were collected, dialyzed in 10 mM glycine, and lyophilized (see FIG. 5).
7) 세파크릴 S-100 겔 여과 크로마토그라피 (Sephacryl S-100 gel filtration chromatography)7) Sephacryl S-100 gel filtration chromatography (Sephacryl S-100 gel filtration chromatography)
Q-세파로즈 FF 음이온 교환 크로마토그라피를 통하여 순수하게 정제된 hGH 에서 극미량으로 잔존이 가능한 내독소 등의 불순물 및 hGH 다이머 (dimer)를 제거하기 위하여 겔 여과를 수행하였다. 동결건조된 hGH 에 증류수를 가하여 만든 20mL 미만 부피의 hGH 용액을 20mM 글라이신으로 미리 평형화된 세파크릴 S-100 칼럼 (26/100, Pharmacia, Sweden)에 로딩하고 유속 4mL/분으로 용출을 수행하였다.Gel filtration was performed to remove impurities such as endotoxins and hGH dimer which can remain in trace amounts in pure purified hGH through Q-Sepharose FF anion exchange chromatography. A less than 20 mL volume of hGH solution made by adding distilled water to lyophilized hGH was loaded onto a pre-equilibrated Sepakril S-100 column (26/100, Pharmacia, Sweden) with 20 mM glycine and elution was carried out at a flow rate of 4 mL / min.
발현 벡터 pT2GH 를 이용한 대장균에서의 hGH 의 생산은 LB 배지를 이용한 배치 배양 및 고농도 배양 각각에서 발현 양상에 차이가 있는데, 배치 배양에서는 T2GH가 가용성과 불용성 (soluble/insoluble)의 비율이 거의 50/50으로 발현되는 반면 고농도 배양에서는 초기에는 가용성으로 발현되나 점차 세포내에 침전되어 주로 불용성으로 생산되었다.Production of hGH in Escherichia coli using the expression vector pT2GH differs in expression pattern in each of batch culture and high concentration culture using LB medium. In the batch culture, the ratio of soluble and insoluble T2GH is almost 50/50 Whereas in the high concentration culture, it was initially expressed as soluble but gradually precipitated in the cells and produced mainly insoluble.
일반적으로 대장균 내에서의 목적 단백질의 발현은 LB 배지를 이용한 회분배양 및 고농도 배양에서 거의 비슷한 경향을 보이는 이유로 해서 본 발명자는 먼저 회분배양을 통하여 가용성 T2GH 로부터 hGH 를 정제하는 과정을 확립하고, 이를 그대로 고농도 배양을 통한 대량 정제에 응용하고자 하였다. 그러나 고농도 배양에서 T2GH 가 주로 불용성으로 생산되고, 이 불용성 침전체는 알칼리용액에서 쉽게 용해되는 특징을 보여 알칼리용해 단계를 이용하였다.In general, the expression of the target protein in E. coli is almost similar in the ash culture and the high concentration culture using the LB medium. Therefore, the present inventor firstly established the process of purifying hGH from soluble T2GH through ash culture, And to apply it to mass purification through high concentration culture. However, T2GH was mainly produced insoluble in high concentration culture, and this insoluble precipitate was easily dissolved in an alkali solution, and an alkali dissolution step was used.
알칼리용해후 크로마토그라피로 정제한 후 엔테로키나제 절단을 행하여 융합단백질로부터 인성장호르몬을 분리시켰다. 반응이 끝난 후 이온교환 수지 및 겔 여과 크로마토그라피를 이용하여 인성장호르몬을 정제하였다.After alkaline dissolution, purification was performed by chromatography, and then enterokinase cleavage was performed to isolate phosphorylcholine from the fusion protein. After the reaction, phosphorus-containing hormones were purified using ion exchange resin and gel filtration chromatography.
〈실시예 6〉 정제된 인성장호르몬의 특성<Example 6> Characterization of purified human growth hormone
1) SDS-PAGE 분석1) SDS-PAGE analysis
먼저 14% 노벡스 프리캐스트(Novex precast) SDS-PAGE 겔 (1.5mm×15웰)을 전기영동 키트에 장착하고 완충액 (0.05M 트리스-HCl, pH 8.4, 0.38M 글리신, 0.1% SDS)을 키트의 챔버에 채운 다음 시료를 2X SDS 완충액 (0.0625M 트리스-HCl, pH6.8, 1% SDS, 15% 글리세롤, 15mM DTT)과 1 : 1 로 섞어 100℃에서 1분간 끓였다. 끓인 시료는 겔의 웰에 로딩하고 125V 에서 2시간 동안 시료를 전개한 다음 겔을 쿠마시 염색 방법으로 염색하였다. 쿠마시 염색방법은 겔을 염색 용액 (0.025% Coomassie blue R-250, 40% 메탄올, 7% 아세트산)에서 30분간 염색한 다음 탈색용액 I (50% 메탄올, 10% 아세트산)에서 1시간, 탈색용액 Ⅱ (5% 메탄올, 7% 아세트산)에서 4시간 이상 탈색하였다.First, a 14% Novex precast SDS-PAGE gel (1.5 mm x 15 well) was mounted in an electrophoresis kit and a buffer (0.05 M Tris-HCl, pH 8.4, 0.38 M glycine, 0.1% SDS) And then the sample was mixed with 1: 1 with 2X SDS buffer (0.0625M Tris-HCl, pH 6.8, 1% SDS, 15% glycerol, 15 mM DTT) and boiled at 100 ° C for 1 minute. The boiled sample was loaded into the well of the gel, and the sample was developed at 125 V for 2 hours, and then the gel was stained with Coomassie staining method. For Coomassie staining, the gel was stained in a staining solution (0.025% Coomassie blue R-250, 40% methanol, 7% acetic acid) for 30 minutes, then decolorized solution I (50% methanol, 10% acetic acid) Ⅱ (5% methanol, 7% acetic acid) for 4 hours or more.
정제된 hGH 는 약 21.5kDa 의 분자량을 가지며 국내시판품 (유트로핀, Lucky Co., 재조합 단백질) 및 시그마사 제품 (인간 뇌하수체 유래)과 비교하였을 때에 동일한 위치에서 단일띠로 나타났다 (도 6 참조).The purified hGH has a molecular weight of about 21.5 kDa and appears as a single band at the same position when compared to domestic commercial products (eutrophin, Lucky Co., recombinant protein) and Sigma products (derived from human pituitary gland).
2) HPLC 분석2) HPLC analysis
먼저 용매 A (10% 아세토니트릴)와 용매 B (80% 아세토니트릴)를 각각 준비한 다음 나일론막 (0.2μm)을 이용하여 진공펌프로 여과하며 가스를 빼내고 용매 A 와 용매 B 에 각각 트리플루오르아세트산 (trifluoroacetic acid, TFA)을 0.05% 되게 가하였다. HPLC (Hitatch HPLC system)에 칼럼 (Vydac C18 reverse phase column, 4.6×250mm)을 연결하고 용매 A로 평형화시켰다. 평형화된 칼럼에 시료를 주입하여 유속 1mL/분, 10-100% 용매 B의 선형 농도구배로 30분동안 시료를 용출하고 215nm에서 흡광도를 측정하여 용출되는 양상을 분석하였다.First, a solvent A (10% acetonitrile) and a solvent B (80% acetonitrile) were prepared. Then, the mixture was filtered with a vacuum pump using a nylon membrane (0.2 μm), gas was withdrawn and trifluoroacetic acid trifluoroacetic acid, TFA) was added in an amount of 0.05%. A column (Vydac C18 reverse phase column, 4.6 x 250 mm) was connected to HPLC (Hitatch HPLC system) and equilibrated with solvent A. The sample was injected into the equilibrated column to elute the sample at a flow rate of 1 mL / min, a linear concentration gradient of 10-100% solvent B for 30 minutes, and the absorbance at 215 nm was measured to analyze the elution pattern.
hGH 는 C18 역상 컬럼에서 약 82% 아세토니트릴 농도에서 용출되었으며, 국내시판품 (유트로핀, Lucky Co.) 및 시그마사 제품과 비교하였을 때에 동일한 위치에서 단일 피크로 나타났다 (도 7 참조).hGH was eluted at a concentration of about 82% acetonitrile in a C18 reversed phase column and showed a single peak at the same position when compared to domestic commercial products (Urotopin, Lucky Co. and Sigma) (see FIG. 7).
3) 펩타이드 맵핑: 트립신, Glu-C (Peptide mapping : trypsin, Glu-C)3) Peptide mapping: trypsin, Glu-C (peptide mapping: trypsin, Glu-C)
hGH 를 40μg 을 포함하는 반응 완충용액 (40mM CHES, pH 9.2)에 원료의 1/20(w/w)에 해당하는 트립신 (TPCK treated, Sigma)을 가한 후 37℃에서 48시간 동안 방치하였다. 반응이 완료된 반응액은 다음과 같은 방법으로 HPLC 를 수행하여 분석하였다.Trypsin (TPCK treated, Sigma) corresponding to 1/20 (w / w) of the raw material was added to a reaction buffer solution (40 mM CHES, pH 9.2) containing 40 μg of hGH and left at 37 ° C for 48 hours. The reaction solution was analyzed by HPLC in the following manner.
먼저 용매 A (10% 아세토니트릴)와 용매 B (80% 아세토니트릴)를 각각 준비한 다음 나일론막 (0.2μm)을 이용하여 진공펌프로 여과하며 가스를 빼내고 용매 A와 용매 B에 각각 트리플루오르아세트산 (TFA)를 0.05% 되도록 가하였다. HPLC (Hitatch HPLC system)에 칼럼 (Vydac C18 reverse phase column, 4.6×250mm)을 연결하고 용매 A로 평형화하였다. 평형화된 칼럼에 시료를 주입하여 유속 1mL/분, 0-90% 용매 B의 선형 농도구배로 30분동안 시료를 용출하며 215nm 에서 흡광도를 측정하여 용출되는 양상이 표준품과 동일한지를 확인하였다.First, a solvent A (10% acetonitrile) and a solvent B (80% acetonitrile) were prepared. Then, the mixture was filtered with a vacuum pump using a nylon membrane (0.2 μm), gas was withdrawn and trifluoroacetic acid TFA) to 0.05%. A column (Vydac C18 reverse phase column, 4.6 x 250 mm) was connected to HPLC (Hitatch HPLC system) and equilibrated with solvent A. The sample was injected into the equilibrated column to elute the sample at a flow rate of 1 mL / min, a linear gradient of 0-90% solvent B for 30 minutes, and the absorbance at 215 nm was measured to confirm that the elution pattern was the same as the standard.
Glu-C (Sigma, U.S.A)를 이용한 펩타이드 맵핑은 50mM 암모늄 바이카보네이트(ammonium bicarbonate, pH 7.8)을 반응액으로 하여 반응 및 HPLC 를 동일한 방법으로 수행하였다.The peptide mapping using Glu-C (Sigma, U.S.A) was carried out by the same method as the reaction and HPLC using 50 mM ammonium bicarbonate (pH 7.8) as a reaction solution.
hGH 를 트립신으로 처리하였을 때에 HPLC 양상이 표준품으로 사용한 유트로핀과 동일함을 보였다 (도 7 참조).When the hGH was treated with trypsin, the HPLC pattern was the same as that of the urotropin used as a standard (see FIG. 7).
4) 면역화학 측정법 (Radioimmunoassay, RIA)4) Radioimmunoassay (RIA)
hGH 의 면역화학 측정은 GH 키트 (Daiichi, Japan)를 이용하였다.GH kit (Daiichi, Japan) was used for the immunochemical measurement of hGH.
먼저 표준품 (standard hGH, Daiichi, Japan)과 국내에 시판되는 재조합 hGH (Lucky Co., 한국) 및 정제된 hGH 를 각각 0-50ng/mL 의 농도로 0.1mL 씩 준비하고, 여기에 표지된125I-hGH 항체용액 (8.1 μCi/10.5mL)을 0.1mL 가하여 준 후, 각각의 반응액에 GH 항체비드를 넣고 15-30℃에서 3시간 동안 200rpm으로 흔들어주었다. 반응이 끝난 후, GH 항체비드를 정제수로 3회 세척하고 방사능을 측정하였다. 그 결과 유트로핀 및 시그마사 제품에 비해 5-10% 범위내에서 더 높게 나타나 순도와 구조적 동질성이 더 우수한 것으로 나타났다.First, standard (standard hGH, Daiichi, Japan) and recombinant hGH is commercially available in Korea (Lucky Co., Korea), and the preparation by 0.1mL of purified hGH in each 0-50ng / mL and the concentration of, here labeled 125 I -hGH antibody solution (8.1 μCi / 10.5 mL) was added to each reaction solution. GH antibody beads were added to each reaction solution, and the mixture was shaken at 15-30 ° C. for 3 hours at 200 rpm. After the reaction, the GH antibody beads were washed three times with purified water and the radioactivity was measured. As a result, higher purity and structural homogeneity were shown in the range of 5-10% than those of eutropin and sigma products.
5) 수용체 결합분석 (Receptor binding assay)5) Receptor binding assay
hGH 의 인간 림프구 세포주인 IM9 에 존재하는 hGH 수용체에 대한 결합분석은 국내에 시판되는 재조합 hGH (Lucky Co., 한국)에 방사성 동위원소를 표지하여 수행하였다.Analysis of the binding of hGH to the hGH receptor present in IM9, a human lymphocyte cell line, was carried out by labeling radioactive isotopes in recombinant hGH (Lucky Co., Korea) marketed in Korea.
Na125I를 재조합 hGH 에 표지하기 위하여는 산화제로서 이오도젠 (Iodogen , PIERCE, U.S.A)을 사용하였는데, 표지된125I-hGH 의 방사능은 9.8 μCi/μg이었다.In order to label Na 125 I with recombinant hGH, Iodogen (PIERCE, USA) was used as the oxidizing agent, and the radioactivity of the labeled 125 I-hGH was 9.8 μCi / μg.
수용체 결합실험은 기본적으로 밴 오버겐 등 (Van Obberghen, E. et al. (1976) J. Biol. Chem. 251, 6844-6851)이 보고한 hGH 수용체 결합 분석방법에 따라 수행하였다. 먼저 0.02nM 의 표지된 hGH 를 0.1-50000nM 농도의 표지되지 않은 hGH와 섞어 mL 당 2×107세포수의 배양된 IM9 림프구와 함께 30℃에서 90분동안 반응시켰다. 반응후 IM9 림프구를 4℃의 신선한 배지로 4회 반복하여 세척하여 결합하지 않은125I-hGH를 제거하였다. IM9 림프구를 세포용출 완충액 (1% 트리톤 X-100, 10% 글리세롤, 20mM HEPES, pH 7.4)으로 파쇄한 후, 세포에 결합한125I-hGH 의 방사능을 측정하였다.Receptor binding experiments were basically performed according to the hGH receptor binding assay method reported by Van Obergen, E. et al. (1976) J. Biol. Chem. 251, 6844-6851). First, 0.02 nM of labeled hGH was mixed with unlabeled hGH at a concentration of 0.1-50000 nM and incubated at 30 ° C for 90 minutes with 2 × 10 7 cells of cultured IM9 lymphocytes per mL. After the reaction, IM9 lymphocytes were washed 4 times with fresh medium at 4 ° C to remove unbound 125 I-hGH. IM9 lymphocytes were disrupted with a cell eluting buffer (1% Triton X-100, 10% glycerol, 20 mM HEPES, pH 7.4) and the radioactivity of 125 I-hGH bound to the cells was measured.
IM9 림프구 세포를 이용한 수용체 결합분석에서도 다른 분석방법에서와 마찬가지로 표준품과 동일한 수용체 결합력을 보였다 (도 9 참조).The receptor binding assay using IM9 lymphocyte cells showed the same receptor binding ability as in the other assay methods (see FIG. 9).
6) 질량 분석6) Mass spectrometry
hGH 의 계산에 의한 이론적 분자량은 22130.23 이며 ESI 질량분석에 의한 측정값이 22132.67±1.65 으로 나타나 계산치와 측정치가 일치하였다.The theoretical molecular weight by calculation of hGH was 22130.23 and the measured value by ESI mass analysis was 22132.67 ± 1.65.
7) 원편광 이색성 분광법 (circular dichroism, CD) 분석7) Circular dichroism (CD) analysis
hGH 와 이의 전구체인 T2GH 의 원편광 이색성 분광법 분석은 Jasco J-700 CD 분광기를 이용하였다. 분광기는 사용전 d-캄포술폰산 (d-camphorsulfonic acid)로 보정하였으며 실온에서 측정하였다. hGH 의 CD 스펙트럼은 표준품으로 사용한 유트로핀과 같은 패턴을 보여주어 동일한 2차 구조를 가짐을 알 수 있었다 (도 10 참조). 또한 전구체인 T2GH 도 hGH 와 거의 비슷한 CD 스펙트럼을 보여 동일한 2차 구조를 가짐을 알 수 있었다.The circular dichroism spectroscopic analysis of hGH and its precursor, T2GH, was performed using a Jasco J-700 CD spectrometer. The spectrometer was calibrated with d-camphorsulfonic acid before use and measured at room temperature. The CD spectra of hGH showed the same secondary structure as the eutrophin used as the standard product (see Fig. 10). In addition, T2GH, which is a precursor, also showed CD spectra almost similar to hGH, showing the same secondary structure.
상기에서 살펴본 바와 같이, 본 발명은 인간 종양괴사인자-α의 올리고펩타이드 (PSDKP)를 융합파트너로 이용하여 융합된 인성장호르몬을 대장균 내에서 가용성 단백질로서 고수율로 발현시킬 수 있고, 이에 포함된 폴리히스티딘 서열(H10)은 금속 킬레이트 수지 (metal-chelated resin) 및 S-세파로스와 같은 양이온 교환수지 등을 이용하여 상기 융합단백질이 용이하게 정제되게 한다.As described above, the present invention can use the oligo peptide (PSDKP) of human tumor necrosis factor-? As a fusion partner to express the fusion human hormone hormone in a high yield as a soluble protein in E. coli, The polyhistidine sequence (H10) facilitates purification of the fusion protein using a metal-chelated resin and a cation exchange resin such as S-Sepharose.
이외에도 본 발명의 T2GH 단백질은 대장균에서 가용성으로 발현된 다음 세포내에서 서서히 침전되는 특징이 있는데, 이는 본 발명의 특이적인 현상으로 대장균에서 단백질을 발현시킬 때 나타나는 불용성 응집체 (inclusion bodies)와는 달리 T2GH 단백질은 알칼리성 용액에 쉽게 용해된다. 따라서 불용성 응집체는 변성제에 녹인 다음 리폴딩 과정을 거쳐야 되나 본 발명의 T2GH 는 알칼리 용해만으로 공정이 끝나기 때문에 리폴딩의 난점을 극복할 수 있다.In addition, the T2GH protein of the present invention is characterized by being soluble in E. coli and then slowly precipitated in the cell. This is a specific phenomenon of the present invention. Unlike the insoluble inclusion bodies that appear when expressing a protein in E. coli, T2GH protein Are easily dissolved in an alkaline solution. Therefore, the insoluble aggregate must be dissolved in the denaturant and then subjected to a refolding process. However, the T2GH of the present invention can overcome the difficulty of refolding because the process is completed only by alkali dissolution.
또한, 상기 융합단백질에 포함된 엔테로키나제 절단 부위인 D4K 는 T2GH 단백질이 엔테로키나제에 의해 절단되어 천연형 인성장호르몬을 생성하는데 유용하다. 이것은 리더펩타이드 없이 대장균에서 직접 발현시 N-말단에 메치오닌이 첨가되어 천연형 인성장호르몬의 제조가 어려운 점을 극복할 수 있도록 해준다.In addition, D4K, an enterokinase cleavage site contained in the fusion protein, is useful for cleaving the T2GH protein by an enterokinase to produce a natural type growth hormone. This allows methionine to be added to the N-terminus when expressed directly in E. coli without the leader peptide, thus overcoming the difficulties of producing natural type growth hormone.
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| WO2015126077A1 (en) * | 2014-02-19 | 2015-08-27 | 한국표준과학연구원 | Method for production, purification and analysis of stable isotope-labeled protein |
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| WO2005021736A1 (en) * | 2003-09-01 | 2005-03-10 | Kobiotech Co. Ltd. | A method for preparing t-20 peptide by high cell density cultivation of recombinant e. coli containing t-20 peptide coding gene |
| WO2015126077A1 (en) * | 2014-02-19 | 2015-08-27 | 한국표준과학연구원 | Method for production, purification and analysis of stable isotope-labeled protein |
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