KR0180804B1 - Preparation process of parathyroid hormone using recombination expression vector - Google Patents
Preparation process of parathyroid hormone using recombination expression vector Download PDFInfo
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- KR0180804B1 KR0180804B1 KR1019950023572A KR19950023572A KR0180804B1 KR 0180804 B1 KR0180804 B1 KR 0180804B1 KR 1019950023572 A KR1019950023572 A KR 1019950023572A KR 19950023572 A KR19950023572 A KR 19950023572A KR 0180804 B1 KR0180804 B1 KR 0180804B1
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- expression vector
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Abstract
본 발명은 융합파트너로 대장균의 복제개시 억제 단백질(Ici A)의 단편을 함유한 L-arabinose 유도성 벡터에, 유로키나제 절단부위의 뉴클레오티드 서열을 포함한 인간 부갑상선 호르몬의 유전자를 삽입하여 제조한 재조합 발현벡터, 그로부터 형질전환된 제조합 미생물 및 전기 미생물을 L-arabinose가 첨가된 배지에서 배양함으로써 인간 부갑상선 호르몬을 대량으로 제조하는 방법에 관한 것이다. 본 발명에서 제조된 재조합 인간 PTH는 천연형 인가 PTH의 활성을 가지고 있음을 확인하였다. 따라서, 본 발명의 재조합 발현벡터로 형질전환된 미생물을 배양하여 L-arabinose로 발현을 유도함으로써, 천연형 인간 PTH가 지닌 활성을 그대로 유지하고 있는 재조합 인간 PTH를 정확한 유도조절을 통하여 고수율로 제조할 수 있다.The present invention provides a recombinant expression vector prepared by inserting a human parathyroid hormone gene including a nucleotide sequence of a urokinase cleavage site into an L-arabinose inducible vector containing a fragment of E. coli replication initiation inhibitory protein (Ici A) as a fusion partner. The present invention relates to a method for producing human parathyroid hormone in large quantities by culturing the transformed synthesized microorganism and the electric microorganism therefrom in a medium to which L-arabinose is added. Recombinant human PTH prepared in the present invention was confirmed to have the activity of the native type PTH. Therefore, by inducing the expression of L-arabinose by culturing the microorganism transformed with the recombinant expression vector of the present invention, the recombinant human PTH that maintains the activity of the native human PTH as it is produced in high yield through accurate induction control can do.
Description
제1도는 발현벡터 △pMA의 유전자 지도를 나타낸다.1 shows a genetic map of the expression vector ΔpMA.
제2(a)도는 발현벡터 △pMA5S의 유전자 지도를 나타낸다.Figure 2 (a) shows a gene map of the expression vector ΔpMA5S.
제2(b)도는 발현벡터 △pMA3S의 유전자 지도를 나타낸다.Figure 2 (b) shows a gene map of the expression vector ΔpMA3S.
제3도는 인간 부갑상선 호르몬(PTH)의 유전자를 제조하기 위한 올리고머들의 염기서열을 나타낸다.Figure 3 shows the nucleotide sequence of oligomers for producing genes of human parathyroid hormone (PTH).
제4도는 합성된 올리고머들의 연결과정을 나타내는 도식이다.4 is a diagram showing the linking process of the synthesized oligomers.
제5도는 유로키나제 절단부위를 제조하기 위한 올리고머들의 염기서열을 나타낸다.Figure 5 shows the nucleotide sequence of oligomers for preparing urokinase cleavage site.
제6도는 본 발명 인간 PTH의 재조합 발현벡터인 pAI5UP 및 pAI3UP를 구축하는 과정을 도식적으로 나타낸 그림이다.Figure 6 is a diagram showing the process of constructing the pAI5UP and pAI3UP recombinant expression vectors of the human PTH of the present invention.
제7(a)도는 인간 PTH의 재조합 발현벡터인 pAI5UP의 유전자 지도를 나타낸다.Figure 7 (a) shows a genetic map of pAI5UP, a recombinant expression vector of human PTH.
제7(b)도는 인간 PTH의 재조합 발현벡터인 pAI3UP의 유전자 지도를 나타낸다.7 (b) shows a genetic map of pAI3UP, a recombinant expression vector of human PTH.
제8(a)도는 발현벡터 pAI5UP의 유전자 중에서 Ici A/유로키나제 절단부위/PTH에 해당하는 부분의 염기서열을 나타낸다.FIG. 8 (a) shows the nucleotide sequence of a portion corresponding to Ici A / Eurokinase cleavage site / PTH in the gene of the expression vector pAI5UP.
제8(b)도는 발현벡터 pAI3UP의 유전자 중에서 Ici A/유로키나제 절단부위/PTH에 해당하는 부분의 염기서열을 나타낸다.FIG. 8 (b) shows the nucleotide sequence of the part corresponding to Ici A / Eurokinase cleavage site / PTH in the gene of the expression vector pAI3UP.
제9도는 발현벡터 pAI5UP와 pAI3UP로 형질전환된 대장균의 L-arabinose 유도에 의한 융합단백질의 발현양상을 나타내는 SDS-PAGE 사진이다.9 is an SDS-PAGE photograph showing the expression pattern of the fusion protein by L-arabinose induction of E. coli transformed with the expression vectors pAI5UP and pAI3UP.
제10도는 발현벡터 pAI5UP와 pAI3UP로 형질전환된 대장균으로부터 L-arabinose 유도에 의해 발현된 융합단백질을 안티-PTH 항체(anti-PTH antibody)를 이용하여 웨스턴 블롯분석(Western blot analysis)한 결과를 나타내는 사진이다.FIG. 10 shows the results of Western blot analysis of the fusion protein expressed by L-arabinose induction from Escherichia coli transformed with the expression vectors pAI5UP and pAI3UP using an anti-PTH antibody. It is a photograph.
제11도는 발현벡터 pAI5UP와 pAI3UP로 형질전환된 대장균으로부터 생산된 Ici A가 융합된 인간 PTH는 봉입체(inclusion body)를 형성함을 나타내는 SDS-PAGE 사진이다.FIG. 11 is an SDS-PAGE photograph showing that human PTH fused with Ici A produced from E. coli transformed with the expression vectors pAI5UP and pAI3UP forms an inclusion body.
제12(a)도는 Ici A 융합된 인간 PTH가 유로키나제에 의해 절단됨을 나타내는 SDS-PAGE 사진이다.Figure 12 (a) is an SDS-PAGE photograph showing that Ici A fused human PTH is cleaved by urokinase.
제12(b)도는 정제된 인간 PTH를 SDS-PAGE한 결과를 나타내는 사진이다.Figure 12 (b) is a photograph showing the result of SDS-PAGE of purified human PTH.
제13(a)도는 정제된 인간 PTH의 수용체에 대한 결합력 시험 결과를 나타내는 그래프이다.Figure 13 (a) is a graph showing the results of the binding test to the receptor of purified human PTH.
제13(b)도는 정제된 인간 PTH의 세포내 cAMP 생성촉진 시험 결과를 나타내는 그래프이다.Figure 13 (b) is a graph showing the results of the intracellular cAMP production promoting test of purified human PTH.
본 발명은 재조합 발현벡터를 이용한 인간 부갑상선 호르몬의 제조방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 융합파트너로 대장균의 복제개시 억제 단백질(Ici A)의 단편을 함유한 L-arabinose 유도성 벡터에, 유로키나제 절단부위의 뉴클레오티드 서열을 포함한 인간 부갑상선 호르몬의 유전자를 삽입하여 제조한 재조합 발현벡터, 그로부터 형질전환된 재조합 미생물 및 전기 미생물로 L-arabinose가 첨가된 배지에서 배양함으로써 인간 부갑상선 호르몬을 대량으로 제조하는 방법에 관한 것이다.The present invention relates to a method for producing human parathyroid hormone using a recombinant expression vector. More specifically, the present invention provides a fusion partner by inserting a gene of human parathyroid hormone including a nucleotide sequence of a urokinase cleavage site into an L-arabinose inducible vector containing a fragment of E. coli replication initiation inhibitory protein (Ici A). The present invention relates to a method for producing a large amount of human parathyroid hormone by culturing in a medium containing L-arabinose with a recombinant expression vector, a recombinant microorganism transformed therefrom, and an electric microorganism.
골다공증(osteoporosis)은 뼈의 질량이 정상적인 경우보다 휠씬 더 많이 감소하여 뼈의 조직이 약해져서 작은 충격에도 쉽게 뼈가 부러지는 등의 부작용을 나타내는 질병이다. 의학, 생물학 등의 발전에 힘입어 노령인구가 계속 증가하고 있어 골다공증 환자도 계속 늘어날 것이므로, 핵가족화 경향에 따라 혼자 사는 노인의 수가 점점 늘어나는 현 시점에서 골다공증은 커다란 사회문제로까지 대두되고 있다.Osteoporosis is a disease that causes side effects such as bone mass being much more reduced than normal, weakening the tissue of the bone and easily breaking the bone even with a small impact. With the development of medicine and biology, the elderly population continues to increase, and osteoporosis patients will continue to increase. As a result, the number of elderly people living alone is increasing as a result of the nuclear family.
일반적으로, 정상적인 뼈조직의 경우, 뼈조직을 파괴하는 세포인 골흡수세포(osteoclast)와 뼈조직을 생성하는 세포인 골생성세포(ostelast)의 활성이 서로 균형을 이루어 항상 뼈조직을 개조(remodeling)하고 있다. 정상인의 경우에도 나이가 많아짐에 따라 골흡수세포의 기능이 골생성세포의 기능을 능가하게 되어, 골밀도의 전반적인 감소현상이 나타나는데, 골다공증 환자의 경우는 이러한 골흡수세포와 골생성세포간의 균형파괴가 정상인에 비하여 휠씬 더 심하다.In general, in normal bone tissue, bone resorption cells (osteoclast), which destroys bone tissue, and osteogenic cells (ostelast), which produce bone tissue, are balanced with each other to always remodel bone tissue. ) Even in normal people, as the age increases, the function of bone-absorbing cells surpasses that of bone-forming cells, resulting in a general decrease in bone density. In osteoporosis patients, the balance between bone-absorbing cells and bone-forming cells It is much worse than normal people.
골흡수세포와 골생성세포간의 균형이 파괴되는 원인에 대하여는 명확하게 알려져 있지 않으나, 폐경기후의 여성들에게 많이 나타나는 타잎 1골다공증의 경우에는 폐경후 일어나는 여성호르몬인 에스트로젠(estrogen)의 분비감소에 그 원인이 있는 것으로 알려져 있다. 따라서, 타잎 1골다공증 치료에는 에스트로젠을 투여하고 있으나, 에스트로젠 투여시 유방암, 자궁내막암 등의 발병률이 높아지는 등의 부작용이 있어, 많은 환자들이 에스트로젠 사용을 꺼리고 있는 실정이다. 또한, 타잎 1골다공증과는 다른 원인에 의해 발명되는 것으로 알려진 타잎 2골다공증에는 에스트로젠의 사용으로 치료효과를 볼 수도 없다.The cause of the disruption of the balance between bone-absorbing cells and bone-forming cells is not known.However, in the case of type 1 osteoporosis, which occurs frequently in postmenopausal women, the cause of postmenopausal female hormone estrogen decreases. It is known. Therefore, the treatment of type 1 osteoporosis has been administered estrogen, but the side effects such as increased incidence of breast cancer, endometrial cancer, etc. when the estrogen administration, many patients are reluctant to use estrogen. In addition, type 2 osteoporosis, which is known to be invented by a cause different from type 1 osteoporosis, has no therapeutic effect by the use of estrogen.
전기 타잎 1골다공증의 치료제인 에스트로젠이 지닌 단점을 보완하고, 에스트로젠의 치료효과를 얻을 수 없는 타잎 2골다공증의 치료제로, 골흡수세포의 기능을 억제하여 뼈조직의 재흡수(resorption)를 저해하는 작용을 나타내는 칼시토닌(calcitonin)이 사용되고 있다. 그러나, 에스트로젠이나 칼시토닌 모두 이미 손실된 뼈질량을 증가시켜 주는 효과도 없고, 다만 골밀도가 더 이상 감소되지 않도록 예방하는 기능만을 갖고 있기 때문에, 효과적인 골다공증 치료제로는 부적당하다.Complements the shortcomings of estrogen, which is a treatment for electric type 1 osteoporosis, and prevents the resorption of bone tissue by inhibiting the function of bone resorption cells. Calcitonin (calcitonin) is used. However, both estrogen and calcitonin have no effect of increasing the lost bone mass, but only have the function of preventing the bone density from being reduced anymore, and thus are not suitable as an effective treatment for osteoporosis.
따라서, 최근에는 부갑상선 호르몬(parathyroid hormone: PTH)이 골밀도 감소의 예방 뿐만 아니라, 골밀도 증대 효과도 초래하며 그와 더불어 부작용이 보고된 바 없어, 골다공증의 우수한 치료제로서 주목받고 있다. 부갑상선 호르몬은 부갑상선의 주세포에서 생성된 아미노산 115개로 구성된 preproPTH(preproparathyroid hormone)가 소포체를 거치면서 절단되어 아미노산 92개로 된 펩타이드 proPTH로 변형되고, 이어 골지(Golgi) 기관을 거치면서 또 다시 절단되어 아미노산 84개로 이루어진 성숙한(mature) PTH로 되는 일련의 과정을 거쳐 합성되고, 혈중으로 분비된 다음, 표적기관인 뼈와 신장으로 이동되는데, 분비된 반감기는 18분에 불과하다.Therefore, in recent years, parathyroid hormone (PTH) not only prevents the reduction of bone density, but also causes the effect of increasing the bone density, and side effects have not been reported, and has been attracting attention as an excellent treatment for osteoporosis. Parathyroid hormone is cleaved through the endoplasmic reticulum preproPTH (preproparathyroid hormone), which consists of 115 amino acids, transformed into a peptide of 92 amino acids, followed by the Golgi organ. It is synthesized through a series of processes of mature PTH consisting of dogs, secreted into the blood, and then transported to target organs, bones and kidneys. The secreted half-life is only 18 minutes.
PTH는 골세포막의 Ca2+펌프를 활성화시켜 골액내 CaHPO4를 외액으로 운반케함으로써, 수분내에 혈중 Ca2+농도를 증가시킨다. 아울러, PTH가 계속 분비되면, 전기 호르몬이 이미 형성되어 있는 골흡수세포를 활성화시키고 골흡수세포의 신생도 자극하여 골생성세포의 활동을 일시적으로 억압함으로써, 골에의 Ca2+침착을 억제하고 Ca2+의 유리(release)를 촉진하여, 혈중으로의 Ca2+과 PO4 3-분비를 증가시키기도 한다. 한편, PTH는 혈중 Ca2+농도에 의한 되먹이기(feedback) 기전으로 분비가 조절된다. 즉, 혈중 Ca2+농도가 단시간내에 10% 감소하면 PTH의 분비는 2배로 증가하지만, 장기간에 걸쳐 Ca2+농도가 낮은 상황에서는 1%의 Ca2+농도 감소에 PTH의 분비는 2배로 늘어나는 강력한 되먹이기 기전으로 조절되고 있다.PTH activates the Ca 2+ pump in the bone cell membrane to deliver CaHPO 4 in bone fluid to the external fluid, thereby increasing blood Ca 2+ concentration in minutes. In addition, if PTH continues to be secreted, it activates bone-absorbing cells that have already formed electrical hormones, stimulates the formation of bone-absorbing cells, and temporarily suppresses the activity of bone-forming cells, thereby inhibiting Ca 2+ deposition in bone. by facilitating the free (release) of the Ca 2+, and also increases the Ca 2+ and PO 4 3- secretion into the blood. On the other hand, PTH secretion is regulated by a feedback mechanism by blood Ca 2+ concentration. In other words, if blood Ca 2+ concentration decreases by 10% within a short time, PTH secretion doubles, but when Ca 2+ concentration is low over a long period, 1% Ca 2+ concentration decreases and PTH secretion doubles. It is being controlled by a powerful feedback mechanism.
이와같은 PTH의 생체내 조절기능과는 달리, PTH를 외부에서 간헐적으로 소량식 투여할 경우 골 형성을 촉진시키는 것으로 보고되었는데(참조: Tam, C.S. et al., Endocrinology, 110:506-512(1982)), 이러한 골 형성 촉진기능이 바로 PTH를 골다공증 치료제로 이용할 수 있는 근거가 되고 있다. PTH의 골 형성 촉진기능에 대한 정확한 기전은 규명되어 있지 않았으나, 투여된 PTH에 의해 생체내 PTH 분비의 억제, 골형성세포에 대한 직접적인 기능 촉진, TGF-β등의 사이토카인(cytokine)을 통한 간접적인 골형성 촉진등의 가설들이 제시되고 있다.In contrast to such in vivo regulation of PTH, intermittent small doses of PTH have been reported to promote bone formation (Tam, CS et al., Endocrinology, 110: 506-512 (1982). The bone formation promoting function is the basis for using PTH as a therapeutic agent for osteoporosis. The exact mechanism of PTH's bone formation promoting function has not been elucidated, but the PTH administered in vivo inhibits PTH secretion in vivo, promotes direct function on osteoblasts, and indirectly through cytokines such as TGF-β. Hypotheses such as phosphorus promotion are suggested.
그러나, 이러한 PTH를 이용하여 골다공증을 치료하기 위해서는 PTH를 장기간 투여하여야 하는데, 지금까지 제조된 PTH는 장기간 투여에 요구되는 PTH의 대량생산을 만족시키지 못하였으므로, 골다공증의 치료에 많은 어려움이 있었다. 이에, 본 발명의 발명자들은 유전공학적 기법을 이용한 재조합 미생물로부터 PTH를 대량 제조하려는 연구를 진행하면서, PTH 아미노 말단의 Ser-Val-Ser 아미노산 서열이 PTH의 생물학적 활성에 필수적인 인자로 알려져 있으므로, 단백질 발현의 숙주로 널리 사용되고 있는 대장균에서의 PTH 발현시 아미노 말단의 메티오닌 제거에 중점을 두었다.However, in order to treat osteoporosis using such PTH, PTH should be administered for a long time. Thus, since PTH manufactured so far did not satisfy the mass production of PTH required for long-term administration, there were many difficulties in treating osteoporosis. Thus, the inventors of the present invention, while proceeding to mass-produce PTH from recombinant microorganisms using genetic engineering techniques, since the Ser-Val-Ser amino acid sequence of the PTH amino terminus is known as an essential factor for the biological activity of PTH, protein expression In PTH expression in E. coli, which is widely used as a host, the focus was on the methionine removal of the amino terminus.
재조합 단백질 발현의 숙주로 널리 사용되고 있는 대장균에는 발현 단백질의 아미노 말단의 번역개시 메티오닌을 제거하는 효소인 메티오닌 특이 아미노 펩티다제가 존재하고 있으나, 외래 단백질이 대장균내에서 대량으로 발현될 때에는 아미노 말단의 메티오닌이 제대로 제거되지 않는 경우가 종종 나타나고 있는데, 이러한 현상은 아미노 말단의 아미노산 서열이 생물학적 활성에 큰 영향을 미치는 PTH와 같은 단백질의 대장균 발현체계를 구축하고자 할 때에는 반드시 해결되어야 할 사항이다.E. coli, which is widely used as a host for recombinant protein expression, contains methionine-specific amino peptidase, an enzyme that removes methionine at the start of translation of the amino terminus of an expressed protein.However, when a large amount of foreign protein is expressed in E. coli, Often, this is not properly removed. This phenomenon must be solved when constructing an E. coli expression system of a protein such as PTH whose amino-terminal amino acid sequence has a great influence on biological activity.
전술한 문제를 해결하기 위하여 주로 다음의 3가지 방법이 이용될 수 있다. 첫째는, 목적단백질의 아미노 말단에 분비 신호서열을 융합시킨 형태로 발현시킴으로써, 목적단백질을 아미노 말단이 절단된(processing) 형태로 대장균의 페리플라즘(periplasm)이나 배양배지로 분비되게 하는 방법인데, 이 방법에는 세포내 활성을 이용하여 성숙된 단백질을 얻을 수 있다는 장점이 있으나, 발현수율이 비교적 낮다는 단점이 있다. 둘째는, 목적단백질을 대장균에서 단독으로 발현시켜, 아미노 말단에 메티오닌이 붙어있는 상태로 대장균으로부터 분리한 다음, 이를 아미노 펩티다제로 절단하여 성숙한 단백질을 얻는 방법인데, 이 방법은 아미노 말단의 메티오닌이 제거된 단백질과 메티오닌이 부착된 단백질 간의 분리 정제가 어렵기 때문에 단백질 정제공정이 복잡하다는 단점을 가지고 있다. 섯째는, 목적단백질을 다른 단백질과의 융합 형태로 발현시켜 융합단백질을 대장균으로부터 분리한 다음, 효소 또는 화학물질을 이용하여 융합단백질로부터 다른 단백질(융합 파트너)을 제거함으로써, 성숙한 목적단백질을 제조하는 방법인데, 이 방법은 아미노 말단의 메티오닌이 제거된 목적단백질을 제공할 뿐만 아니라, 목적단백질의 발현효율도 증대시킨다는 장점을 지니고 있다.In order to solve the above problem, the following three methods can be mainly used. The first method is to express the secreted signal sequence in the form of a fusion to the amino terminus of the target protein, thereby secreting the target protein into the periplasm or culture medium of E. coli in the form of the amino terminus processing. This method has the advantage of obtaining mature protein using intracellular activity, but has a disadvantage of relatively low expression yield. Secondly, the target protein is expressed alone in E. coli, isolated from E. coli with methionine attached to the amino terminus, and then cleaved with amino peptidase to obtain a mature protein. It is difficult to separate and purify between the removed protein and the protein to which methionine is attached. Fifth, the target protein is expressed in the form of fusion with other proteins to isolate the fusion protein from Escherichia coli, and then another enzyme (fusion partner) is removed from the fusion protein using enzymes or chemicals to prepare a mature target protein. This method has the advantage of not only providing the target protein from which the amino-terminal methionine has been removed, but also increasing the expression efficiency of the target protein.
따라서, 본 발명의 발명자들은 재조합 대장균으로부터 아미노 말단인 메티오닌이 제거된 PTH를 대량 제조하기 위하여, 상기 세 번째 방법을 이용하였다. 즉, 융합파트너로 이용되는 대장균의 복제개시 억제 단백질(이하, 'Ici A'라 함)의 유전자 단편을 L-arabinose 유도성 벡터에, 유로키나제에 의한 특이적 절단부위의 유전자 및 대장균에서의 코돈을 사용하도록 제조한 인간 PTH의 유전자를 삽입하여 발현벡터를 제조하고, 대장균을 형질전환시킨 다음, 그로부터 융합단백질을 분리하여 유로키나제로 절단함으로써, 천연형 인간 PTH의 활성을 지닌 재조합 PTH를 대량으로 제조할 수 있었다.Therefore, the inventors of the present invention used the third method to mass-produce PTH free of amino-terminal methionine from recombinant E. coli. In other words, the gene fragment of the E. coli replication initiation inhibitory protein (hereinafter referred to as 'Ici A') used as a fusion partner was transferred to the L-arabinose inducible vector, and the gene of the specific cleavage site by urokinase and codon in E. coli. An expression vector was prepared by inserting a gene of human PTH prepared for use, transformed into E. coli, and then isolated from the fusion protein and cleaved with urokinase, thereby producing a large amount of recombinant PTH having the activity of natural human PTH. Could.
결국, 본 발명의 주된 목적은 Ici A의 단편을 함유한 L-arabinose 유도성 벡터에, 유로키나제에 의한 특이적 절단부위의 뉴클레오티드 서열을 포함한 PTH의 유전자를 삽입하여 제조한 재조합 발현벡터 및 그로부터 형질전환된 재조합 미생물을 제공하는 것이다.After all, the main object of the present invention is a recombinant expression vector prepared by inserting a gene of PTH containing a nucleotide sequence of a specific cleavage site by urokinase into a L-arabinose inducible vector containing a fragment of Ici A and transforming therefrom. To provide a recombinant microorganism.
본 발명의 또 다른 목적은 전기 형질전환된 재조합 미생물을 L-arabinose가 첨가된 배지에서 배양함으로써 인간 PTH를 대량으로 제조하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for producing human PTH in large quantities by culturing the recombinant microorganisms transformed in L-arabinose added medium.
이하, 본 발명을 보다 구체적으로 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
Ici A 유전자의 500bp와 300bp에 해당하는 위치에 각각 SmaI 제한효소 부위를 도입한 발현벡터 △pMA5S 및 △pMA3S로 각각 형질전환된 대장균 MC1061: △pMA5S(KCCM-10072)및 대장균 MC1061: △pMA3S(KCCM-10069)로 부터 전기 발현벡터를 분리하고, 이 발현벡터에 유로키나제에 의한 특이적 절단 부위의 뉴클레오티드 서열을 포함한 인간 PTH 유전자를 삽입하여 융합단백질의 발현벡터를 제조하였다.E. coli MC1061: ΔpMA5S (KCCM-10072) and E. coli MC1061: ΔpMA3S (KCCM) transformed with the expression vectors ΔpMA5S and ΔpMA3S, respectively, which introduced SmaI restriction enzyme sites at positions corresponding to 500 bp and 300 bp of the Ici A gene, respectively. -10069) was isolated, and the expression vector of the fusion protein was prepared by inserting the human PTH gene including the nucleotide sequence of the specific cleavage site by urokinase into the expression vector.
우선, 인간의 PTH 유전자는 천연형 인간 PTH의 아미노산 서열로 번역되며 대장균에서 코돈 사용빈도가 높은 염기서열을 가지도록 제조하였다. 또한, 융합단백질 사이에 유로키나제에 의해 특이적으로 절단되는 아미노산 서열을, 가장 바람직하게는 -Thr-Gly-Arg을 포함하도록 유로키나제 절단부위(참조: 대한민국 특허출원 제95-19208호)를 합성하여, 인간 PTH 유전자 앞에 위치하도록 하였다.First, the human PTH gene is translated into the amino acid sequence of native human PTH and prepared to have a high sequence of codon usage in E. coli. In addition, a kinase cleavage site (see Korean Patent Application No. 95-19208) is synthesized to include an amino acid sequence specifically cleaved by urokinase between fusion proteins, most preferably -Thr-Gly-Arg. It was placed before the human PTH gene.
상기 발현벡터 △pMA5S와 △pMA3에 유로키나제 절단부위를 포함하는 인간 PTH 유전자를 삽입하여 융합단백질의 발현벡터 pAI5UP 및 pAI3UP를 각각 제조하고, 그의 형질전환체를 배향하여 L-arabinose로 발현을 유도함으로써, Ici A가 융합된 인간 PTH가 대량 발현됨을 확인하였다. 따라서, 상기 발현벡터 △pMA5S와 △pMA3S에 인간 PTH 유전자를 삽입하여 제조한 pAI5UP 및 pAI3UP은 기존의 어떠한 발현벡터보다도 높은 인간 PTH의 발현율 및 정확한 유도조절을 나타냄을 알 수 있었다.By inserting the human PTH gene including a urokinase cleavage site into the expression vectors ΔpMA5S and ΔpMA3, expression vectors pAI5UP and pAI3UP of the fusion protein were prepared, and the transformants were oriented to induce expression by L-arabinose. It was confirmed that a large amount of human PTH fused with Ici A. Therefore, it was found that pAI5UP and pAI3UP prepared by inserting the human PTH gene into the expression vectors ΔpMA5S and ΔpMA3S showed higher expression rate and precise induction regulation of human PTH than any conventional expression vector.
아울러, 상기에서 발현된 Ici A가 융합된 인간 PTH는 형질전환체 내에서 봉입체(inclusion body) 형태로 분리됨을 확인하고, 봉입체를 수득하여 유로키나제를 처리하고 Ici A/PTH 융합단백질로부터 재조합 인간 PTH를 분리 및 정제하였다.In addition, it was confirmed that the human PTH fused with Ici A expressed above was separated into an inclusion body in a transformant, and the inclusion body was obtained to treat urokinase and to obtain recombinant human PTH from Ici A / PTH fusion protein. Isolate and purify.
한편, 생체내에서 PTH는 콩팥 및 무기질화한 골로부터 칼슘의 재흡수를 촉진하여, 혈중 칼슘농도를 증대시킴으로써 칼슘의 항상성을 조절하는 활성을 가지고 있는데, 전기 활성은 PTH가 골세포나 콩팥세포의 표면에 있는 고친화성 수용체에 결합하여 수용체와 연관되어 있는 아데닐레이트 사이 클레이즈(adenylate cyclase)를 활성화시킴으로써, ATP로부터 형성된 세포내 이차 신호전달 물질인 cAMP에 의해 매개되는 것으로 보고되고 있다(참조: Donahue, H.J. et al., Endocrinology, 126: 1471-1477(1990)). 이러한 보고와 관련하여, 본 발명자들은 상기에서 정제된 재조합 인간 PTH가 천연 PTH의 활성을 가지고 있는지 확인하기 위하여, 골세포 또는 콩팥세포에 존재하는 수용체(receptor)에 대한 PTH의 결합력과 세포내 cAMP(cyclic AMP) 형성 촉진정도를 측정하였다. 그 결과, 상기에서 제조된 재조합 인간 PTH는 수용체 결합력 및 세포내 cAMP 생성 촉진능력을 가지고 있음을 확인하였다.On the other hand, in vivo, PTH has the activity of regulating the homeostasis of calcium by promoting calcium resorption from kidneys and mineralized bone and increasing blood calcium concentration. It has been reported to be mediated by cAMP, an intracellular secondary signaling material formed from ATP by binding to a high affinity receptor at and activating the adenylate cyclase associated with the receptor (Donahue). , HJ et al., Endocrinology, 126: 1471-1477 (1990)). In connection with this report, the present inventors, in order to determine whether the recombinant human PTH purified above has the activity of native PTH, the binding capacity of PTH to receptors present in bone cells or kidney cells and intracellular cAMP ( Cyclic AMP) formation promotion was measured. As a result, it was confirmed that the recombinant human PTH prepared above had receptor binding capacity and intracellular cAMP production promoting ability.
따라서, 본 발명의 재조합 발현벡터 pAI5UP 또는 pAI3UP로 형질전환된 미생물을 배양하여 L-arabinose로 발현을 유도함으로써, 천연형 인간 PTH가 지닌 활성을 그대로 유지하고 있는 재조합 인간 PTH를 정확한 유도조절을 통하여 고수율을 제조할 수 있다.Therefore, by inducing the expression of L-arabinose by culturing the microorganism transformed with the recombinant expression vector pAI5UP or pAI3UP of the present invention, the recombinant human PTH that maintains the activity of the native human PTH as it is through high induction regulation Yields can be prepared.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
[실시예 1]Example 1
발현벡터 △pMA의 제조Preparation of Expression Vector ΔpMA
살모넬라 티피무리움(Salmonella typhymurium) LT2 균주에서 염색체 DNA를 추출하고 이를 EcoRI 제한효소를 처리한 다음, pUC19 벡터에 삽입하여 pUC-살모넬라 라이브러리를 제조하였다. pUC-살모넬라 라이브러리를 대장균 DH5α균주(E. coli DH5α, F' endAl hsdR17(rk-mk+) supE44 thi-1 recAl gyrA(NaF) U169△(lacZAY-argF) deoR)에 도입시켜, 형질전환된 대장균 콜로니들을 얻었다. 한편, araB-C 조절부위와 araC 단백질의 아미노말단(N-terminal) 3번째 아미노산(Glu)까지를 포함할 수 있는 염기서열에 대한 15mer인 5'-GCCATCGTCTTACTC-3'와 14mer인 5'-GCGTTTCAGCCATG-3' 올리고뉴클레오티드를 합성하였다. 이들을 탑침자(probe)로 사용하며, 콜로니 혼성화(colony hybridization)를 실시하여 전기에서 제조된 pUC-살모넬라 라이브러리로부터 araB-A 및 araC 유전자를 포함하는 클론을 선별하였다.Chromosomal DNA was extracted from Salmonella typhymurium LT2 strain, treated with EcoRI restriction enzyme, and inserted into pUC19 vector to prepare pUC-Salmonella library. pUC-Salmonella library was introduced into the E. coli DH5α strain (E. coli DH5α, F 'endAl hsdR17 (rk - mk + ) supE44 thi-1 recAl gyrA (NaF) U169Δ (lacZAY-argF) deoR), Got colonies. Meanwhile, 5'-GCCATCGTCTTACTC-3 ', which is 15mer, and 5'-GCGTTTCAGCCATG, which are 15mers for the nucleotide sequence which may include the araB-C regulatory region and the amino acid (N-terminal) third amino acid (Glu) of the araC protein -3 'oligonucleotide was synthesized. These were used as probes and colony hybridization was performed to select clones containing the araB-A and araC genes from the previously prepared pUC-Salmonella library.
이렇게 선별된 플라스미드 pUC19-ara에 AvaI/SalI 제한효소를 처리하여 얻은 2.52kb의 DNA 단편; 및 , pUC119(Maniatis et al., Molecular Cloning 2nd ed., 1989) 벡터에 HindIII/SalI 제한효소를 처리하여 얻은 3.18kb의 DNA 단편을 T4DNA 연결효소로 연결하여, 5.7kb의 pUC119-araBC 벡터를 제조하였다. 이로부터 단일가닥의 DNA를 얻은 다음, araB 프로모터의 하단에 위치하는 araB 단백질 구조유전자의 번역개시부호에 NdeI 제한효소가 자르는 염기서열인 5'-CATATG-3'가 삽입되고, araB 프로모터의 샤인-달가노 염기서열이 5'-TAAGGAGG-3'으로 변형되도록, 부위특이적 돌연변이를 야기시켰다.A 2.52 kb DNA fragment obtained by treating AvaI / SalI restriction enzyme with the selected plasmid pUC19-ara; And, pUC119 (Maniatis et al., Molecular Cloning 2nd ed., 1989) of the 3.18kb DNA fragment obtained by treating a HindIII / SalI restriction enzymes and the vector through to T 4 DNA ligase, the 5.7kb vector pUC119-araBC Was prepared. After obtaining single-stranded DNA, 5'-CATATG-3 ', a nucleotide sequence cut by NdeI restriction enzyme, is inserted into the translation initiation code of the araB protein structural gene located at the bottom of the araB promoter, and the shine- of the araB promoter is inserted. Site specific mutations were caused to modify the Dalgano sequence to 5'-TAAGGAGG-3 '.
이렇게 변형된 ara클론에 다시 EcoRI-PvuII 제한효소를 처리하여, araB-C 유전자를 함유하는 2.61kb의 DNA 단편을 얻었다. 한편, pUC18로부터 유래한 228bp의 다클론부위를 포함한 MpKL10벡터에 NdeI 제한효소를 처리하고, 클레노우 효소로 평활말단화하여 T4DNA 연결효소로 재결합시킨 다음, EcoRI-PvuII 제한효소를 처리하여 2.7kb의 DNA 단편을 얻었다. 전기에서 얻은 2.61kb의 DNA 단편과 2.7kb의 DNA 단편을 연결시킨 다음, NdeI-EcoRI 제한효소 부위의 중간에 NcoI 제한효소 부위를 삽입하여, 약 5.32kb의 발현벡터 △pMA를 제조하였다(참조: 대한민국 특허출원 제 94-11449호). 이렇게 제조된 △pMA의 유전자 지도를 제1도에 나타내었다.The modified ara clone was further treated with EcoRI-PvuII restriction enzyme to obtain a 2.61 kb DNA fragment containing the araB-C gene. On the other hand, NdeI restriction enzyme was treated to MpKL10 vector containing 228bp polyclonal region derived from pUC18, blunt-terminated with Klenow enzyme, recombined with T 4 DNA ligase, and then treated with EcoRI-PvuII restriction enzyme. A DNA fragment of kb was obtained. A 2.61 kb DNA fragment and a 2.7 kb DNA fragment were linked to each other, and an NcoI restriction enzyme site was inserted in the middle of the NdeI-EcoRI restriction enzyme site to prepare an expression vector ΔpMA of about 5.32 kb. Korean Patent Application No. 94-11449). The gene map of ΔpMA thus prepared is shown in FIG.
[실시예 2]Example 2
융합 파트너인 Ici A 유전자의 제조Preparation of Ici A Gene, a Fusion Partner
대장균 DH5 α 균주로부터 염색체 DNA를 분리한 다음, 중합효소 연쇄반응(polymerase chain reaction: PCR)을 수행하고 Ici A 유전자를 증폭하여 수득한 약 900bp에 해당하는 Ici A 유전자를 실시예 1에서 제조한 △pMA의 NdeI/EcoRI 제한효소 절단부위에 삽입하여 Ici A 발현벡터 △pMA5를 제조하였다. 이 발현벡터로 형질전환된 대장균으로부터 분자량이 33kDa인 Ici A 단백질이 발현됨을 확인하였다. 그러나, 전기에서 발현된 크기가 큰 Ici A 단백질과 외래 단백질의 융합시, 발현된 목적단백질의 최종 생산량이 적어질 단점이 있으므로, Ici A 유전자의 크기를 줄이면서 발현율은 감소시키지 않는 적당한 크기의 융합 파트너 Ici A 유전자를 제조하기 위하여, Ici A 유전자의 500bp와 300bp에 해당하는 위치에 각각 SmaI 부위를 도입하는 돌연변이를 수행하여 △pMA5S 및 △pMA3S를 제조하였다. 이렇게 제조된 △pMA5S 및 △pMA3S의 유전자 지도를 각각 제2(A) 및 2(B)도에 나타내었다.Chromosome DNA was isolated from the E. coli DH5 α strain, followed by polymerase chain reaction (PCR), and amplified Ici A gene. An Ici A expression vector ΔpMA5 was prepared by inserting the NdeI / EcoRI restriction enzyme cleavage site of pMA. It was confirmed that the Ici A protein having a molecular weight of 33 kDa was expressed from E. coli transformed with the expression vector. However, when fusion of a large-sized Ici A protein and a foreign protein expressed in the past, there is a disadvantage in that the final output of the expressed target protein is reduced, so that the fusion of a suitable size that does not reduce the expression rate while reducing the size of the Ici A gene To prepare the partner Ici A gene, ΔpMA5S and ΔpMA3S were prepared by performing mutations to introduce SmaI sites at positions corresponding to 500 and 300 bp of the Ici A gene, respectively. Gene maps of ΔpMA5S and ΔpMA3S thus prepared are shown in FIGS. 2 (A) and 2 (B), respectively.
전기 발현벡터 △pMA5S 및 △pMA3S로 대장균 MC1061(E. coli MC1061, F-araD139 △(ara-leu)7696 galE15 galk16 △(lac) × 74rpsL(Strr) hsdR2(rk-mk+) mcrA mcrB1)을 형질전환시켜 제조된 미생물(KCCM-10072 및 KCCM-10069)을 배양하여 Ici A 단백질의 발현을 확인한 결과, Ici A 유전자의 500bp와 300bp에 해당하는 위치에 각각 삽입된 SmaI부위는 Ici A 유전자의 발현에 어떠한 영향도 미치지 않고, Ici A 유전자 단편을 융합 파트너로 적절히 이용할 수 있음을 확인하였다(참조: 동일자 출원하는 『대장균의 복제개시 억제 단백질을 융합 파트너로 이용하는 발현벡터』).E. coli MC1061 (E. coli MC1061, F-araD139 Δ (ara-leu) 7696 galE15 galk16 Δ (lac) × 74rpsL (Str r ) hsdR2 (rk − mk + ) mcrA mcrB1) with the expression vectors ΔpMA5S and ΔpMA3S As a result of culturing microorganisms (KCCM-10072 and KCCM-10069) prepared by transformation, the expression of Ici A protein was confirmed. As a result, SmaI sites inserted at positions corresponding to 500 and 300 bp of Ici A gene were expressed in Ici A gene. It has been confirmed that the Ici A gene fragment can be appropriately used as a fusion partner without any effect on the human body (see, `` Expression vector using E. coli replication initiation inhibiting protein as a fusion partner '').
[실시예 3]Example 3
인간 PTH 유전자의 제조Preparation of the Human PTH Gene
천연형 인간 PTH의 아미노산 서열로 번역되며 대장균에서 코돈 사용빈도가 높은 염기서열을 가지는 인간 PTH 유전자를 제조하기 위하여, 먼저 PTH의 센스(sense)와 안티센스(antisense) 가닥에 해당하는 12개의 올리고뉴클레오티드를 합성하였다(참조: 제3도).To prepare a human PTH gene which is translated into the amino acid sequence of a native human PTH and has a high codon sequence in Escherichia coli, 12 oligonucleotides corresponding to the sense and antisense strands of PTH are first prepared. Synthesis (see Figure 3).
전기 12개의 올리고머들을 선택적으로 말단에 인산화(kination), 재결합(annealing), 용출(elution) 및 T4 연결효소를 이용하여 올리고머들을 연결하였다. 먼저, 자체적인 연결을 막기 위하여, 올리고머 #4, #5, #8, #9, #12 및 #13의 말단을 인산화시키고, #3.#4, #5.#6, #7. #8, #9.#10, #11.#12 및 #13.#14을 한쌍으로 하여 재결합시킨 다음, 폴리아크릴아마이드젤 전기영동하여 정확히 재결합된 이중가닥의 올리고머들 Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ 및 Ⅶ만을 용출하였다. 이중가닥의 올리고머 Ⅶ의 3'은 XbaI 제한효소의 절단부위를 가지고 있어, 그 제한효소로 절단하였을 때 접착말단을 형성하므로, 실시예 2에서 제조한 Ici A 융합 발현벡터의 XbaI 부위로 쉽게 클로닝될 수 있다.The twelve oligomers were selectively linked to oligomers using phosphorylation, annealing, elution and T4 ligase at the ends. First, to prevent self-linking, the ends of oligomers # 4, # 5, # 8, # 9, # 12 and # 13 are phosphorylated, and # 3. # 4, # 5. # 6, # 7. Recombination of # 8, # 9. # 10, # 11. # 12, and # 13. # 14 in pairs, followed by polyacrylamide gel electrophoresis to accurately recombine double-stranded oligomers II, III, IV, V , Only VI and X were eluted. 3 ′ of the double-stranded oligomer VII has a cleavage site of XbaI restriction enzyme and forms an adhesive end when cleaved with the restriction enzyme, so that it can be easily cloned into the XbaI site of the Ici A fusion expression vector prepared in Example 2. Can be.
전기에서 용출한 이중가닥 올리고머의 말단을 인산화시킨 후, 이중가닥의 Ⅱ과Ⅲ, Ⅳ와 Ⅴ, Ⅵ과 Ⅶ을 각각 T4 연결효소로 연결한 다음, 폴리아크릴아마이드 젤 전기영동하여 정확히 연결된 Ⅱ/Ⅲ, Ⅳ/Ⅴ 및 Ⅵ/Ⅶ을 용출하였다. 어어, Ⅱ/Ⅲ, Ⅳ/Ⅴ 및 Ⅵ/Ⅶ 3가지 단편의 말단을 인산화시키고 Ⅰ과 Ⅱ/Ⅲ, Ⅳ/Ⅴ 및 Ⅵ/Ⅶ을 T4 연결효소를 이용하여 연결, 또 다시 Ⅰ/Ⅱ/Ⅲ과 Ⅳ/Ⅴ/Ⅵ/Ⅶ를 연결하여, 최종적으로 유로키나제 절단부위를 포함하면서 대장균에서의 코돈 사용빈도가 높은 염기서열을 가지는 인간 PTH 유전자를 합성하였다(참조: 제4도). 제4도에서, 괄호 안의 숫자는 염기의 수를 나타낸다. 상기에서 이중가닥의 올리고머 Ⅰ에 대한 설명은 하기 실시예 4에 상세히 기재되어 있다.Phosphorylate the ends of the double-stranded oligomers eluted in the former, connect the double-stranded II and III, IV and V, VI and V with T4 linkage enzymes, respectively, and then polyacrylamide gel electrophoresis to correctly connect the II / III. , IV / V and VI / v were eluted. Er, phosphorylation of the ends of three fragments II / III, IV / V and VI / VIII, and linking I and II / III, IV / V and VI / VIII using T4 ligase and again I / II / III Finally, human PTH genes were synthesized by linking Ⅳ / V / VI / Ⅶ with a nucleotide sequence that contains a urokinase cleavage site and has high codon usage in E. coli (see FIG. 4). In Figure 4, the numbers in parentheses indicate the number of bases. The description for the double stranded oligomer I above is described in detail in Example 4 below.
[실시예 4]Example 4
유로키나제 절단부위의 합성Synthesis of urokinase cleavage site
융합단백질로부터 PTH를 용이하게 분리하기 위하여, 다음과 같이 유로키나제 절단부위를 합성하였다.In order to easily separate PTH from the fusion protein, a urokinase cleavage site was synthesized as follows.
유로키나제 절단부위에 Gly-Thr-Gly-Arg을 포함하도록 하면서(참조: 대한민국 특허출원 제 95-19208호), 전기 서열 앞에 비교적 작은 분자량의 아미노산을 덧붙여 가변성(flexibility)을 부여하였다. 또한, 5'부분은 어떠한 표적(target) 유전자라도 그의 개방해독틀(open reading frame)에 맞게 용이하게 유합할 수 있도록, 평활말단(blunt-end)으로 시작되는 3개의 제한효소 SmaI, ScaI 및 PuvII 인식부위를 포함하도록 제조하였으며, 유로키나제 절단부위와 인간 PTH의 연결부분에는 BglII 제한효소 부위가 위치하도록 제조하였다. 이러한 조건을 만족시키는 센스 올리고머(34mer) #1과 안티센스 올리고머(34mer) #2를 합성하여(참조: 제5도), 이들을 재결합시킨 다음, 폴리아크릴아마이드 젤 전기영동하여 정확히 재결합된 이중가닥의 올리고머 Ⅰ을 용출하였다. 이중가닥의 올리고머 Ⅰ은 양끝이 모두 평활말단이므로, 재결합하기 전에 #2 올리고머만을 인산화시켜, 올리고머 Ⅰ의 이량체(dimer) 및 삼량체(trimer) 등의 형성을 방지하였다. 이중가닥의 올리고머 Ⅰ이 전기 실시예 3에서 합성된 인간 PTH 유전자 앞에 위치하도록 T4 연결효소를 이용하여 연결하였다(참조: 제4도).While including Gly-Thr-Gly-Arg in the urokinase cleavage site (see Korean Patent Application No. 95-19208), a relatively small molecular weight amino acid was added before the sequence to give flexibility. In addition, the 5 'portion contains three restriction enzymes SmaI, ScaI and PuvII beginning with blunt-ends, allowing any target gene to be easily coalesced into its open reading frame. It was prepared to include a recognition site, and the BglII restriction enzyme site was located at the junction of the urokinase cleavage site and the human PTH. Sense oligomer (34mer) # 1 and antisense oligomer (34mer) # 2 satisfying these conditions were synthesized (see FIG. 5), and then recombined, followed by polyacrylamide gel electrophoresis to accurately recombined double stranded oligomer I eluted. Since the double-stranded oligomer I was smooth at both ends, only the # 2 oligomer was phosphorylated before recombination, thereby preventing the formation of dimers and trimers of oligomer I. The double stranded oligomer I was linked using T4 ligase so as to be located before the human PTH gene synthesized in Example 3 (see FIG. 4).
[실시예 5]Example 5
인간 PTH의 발현벡터 pAI5UP 및 pAI3P의 제조Preparation of Expression Vectors pAI5UP and pAI3P of Human PTH
실시예 2에서 제조된 Ici A 발현벡터 △pMA5S와 △pMA3S를 SmaI/XbaI 제한효소로 절단하여, 각각 500bp와 300bp에 해당하는 Ici A 유전자 단편을 포함하는 발현벡터의 단편을 제조하였다. 전기 실시예 2 및 3에서 합성된 유로키나제 절단부위를 포함하는 인간 PTH 유전자의 양끝이 평활말단과 XbaI의 접착말단인 점을 고려하면, SmaI/XbaI으로 처리된 발현벡터 △pMA5S와 △pMA3S내로 유로키나제 절단부위를 포함하는 인간 PTH 유전자는 쉽게 서브클로닝될 수 있다. 이렇게 하여 제조된 araB 프로모터 체계에 의해 발현조절 가능한 발현벡터를 각각 pAI5UP 및 pAI3UP라 명명하였으며, 전기 각 발현벡터의 구축과정을 제6도에 나타내었다. 제7(A) 및 7(B)도는 발현벡터 pAI5UP 및 pAI3UP의 유전자 지도를 각각 나타내며, 제8(A) 및 8(B)도는 발현벡터 pAI5UP 및 pAI3UP의 유전자 중에서 Ici A/유로키나제 절단부위/PTH에 해당하는 부분의 염기서열을 각각 나타낸다.The Ici A expression vectors ΔpMA5S and ΔpMA3S prepared in Example 2 were digested with SmaI / XbaI restriction enzymes to prepare fragments of expression vectors containing Ici A gene fragments corresponding to 500 bp and 300 bp, respectively. Considering that both ends of the human PTH gene including the urokinase cleavage site synthesized in Examples 2 and 3 are the adhesion end between the smooth end and XbaI, urokinase cleavage into the expression vectors ΔpMA5S and ΔpMA3S treated with SmaI / XbaI. Human PTH genes comprising the site can be easily subcloned. Expression vectors capable of expression control by the araB promoter system thus prepared were named pAI5UP and pAI3UP, respectively, and the construction process of each expression vector was shown in FIG. Figures 7 (A) and 7 (B) show gene maps of expression vectors pAI5UP and pAI3UP, respectively. Figures 8 (A) and 8 (B) show Ici A / Eurokinase cleavage sites / PTH in the genes of expression vectors pAI5UP and pAI3UP. The nucleotide sequence of the part corresponding to each is shown.
[실시예 6]Example 6
재조합 단백질의 발현Expression of Recombinant Protein
실시예 5에서 제조된 발현벡터 pAI5UP 및 pAI3UP로 대장균 MC1061을 각각 형질전환시킨 다음, 각 형질전환체를 대장균 MC1061:pAI5UP 및 대장균 MC1061:pAI3UP라 명명하고, 1995년 7월 6일 국제기탁기관인 사단법인 한국종균협회 부설 미생물보존센타(KCCM)에 기탁번호 KCCM-10071 및 KCCM-10070으로 각각 기탁하였다.E. coli MC1061 was transformed with the expression vectors pAI5UP and pAI3UP prepared in Example 5, and each transformant was named E. coli MC1061: pAI5UP and E. coli MC1061: pAI3UP, and on July 6, 1995, an international depository organization The microbial preservation center (KCCM) attached to the Korea spawn association was deposited with the accession number KCCM-10071 and KCCM-10070 respectively.
전기에서 형질전환된 대장균을 앰피실린을 함유하는 LB평판 배지에서 37℃의 온도조건으로 배양하여, 생장하는 대장균 콜로니를 선별한 다음, 이를 앰피실린이 포함된 LB 액체배양액에 접종하고, 37℃에서 180rpm으로 진탕배양하였다. 그런다음, 배양세포의 농도가 600nm에서 0.5 내지 0.6의 흡광도에 도달했을 때, 1%(w/v)의 L-arabinose를 배양액에 첨가하여 Ici A가 융합된 PTH의 발현을 유도한 후, 세포를 파쇄하고 SDS-PAGE를 수행하였다(참조: 제9도). 제9도에서, 제1 내지 4 레인과 제6 내지 8레인은 L-arabinose로 유도하기 전의 총단백질, 제9 내지 15레인은 제1 내지 4레인과 제6 내지 8레인의 각 형질전환체와 동일하되 L-arabinose로 유도한 후의 총단백질을 각각 나타내며; 제1레인 및 제9레인은 △pMA5로 형진전환된 대장균의 총단백질, 제2 및 3레인과 제10 및 11레인은 pAI3UP로 형질전환된 대장균의 총단백질, 제4 및 8레인과 제12 및 15레인은 pAI5UP로 형질전환된 대장균의 총단백질, 제6레인과 제13레인은 △pMA3S로 형질전환된 대장균의 총단백질, 제7레인과 제14레인은 △pMA5S로 형질전환된 대장균의 총단백질, 제5레인은 단백질 표준 시료물질로서 a는 포스포릴레이즈(phosphorylase B, 106kDa), b는 소혈청 알부민(bovine serum albumin, 80kDa), c는 오발부민(ovalbumin, 49.5 kDa), d는 카보닉 안하이드레이즈(carbonic anhydrase, 32.5kDa), e는 소이빈 드립신 저해제(soybean trypsin inhibitor, 27.5kDa) 및 f는 라이소자임(lysozyme, 18.5kDa)을 각각 나타낸다.The transformed Escherichia coli was cultured in an LB flat medium containing ampicillin at a temperature of 37 ° C. to select the growing E. coli colonies, and then inoculated into the LB liquid culture solution containing ampicillin, at 37 ° C. Shake culture at 180 rpm. Then, when the concentration of the cultured cells reached an absorbance of 0.5 to 0.6 at 600 nm, 1% (w / v) of L-arabinose was added to the culture to induce the expression of Ici A fused PTH. Was disrupted and SDS-PAGE was performed (see Figure 9). In FIG. 9, lanes 1 to 4 and lanes 6 to 8 are total proteins prior to induction of L-arabinose, and lanes 9 to 15 correspond to transformants of lanes 1 to 4 and lanes 6 to 8, respectively. The same but represents the total protein after induction with L-arabinose; Lanes 1 and 9 are total protein of E. coli transformed into ΔpMA5, lanes 2 and 3, and lanes 10 and 11 are total protein of E. coli transformed with pAI3UP, lanes 4 and 8 and 12 and Lane 15 is the total protein of E. coli transformed with pAI5UP, lanes 6 and 13 are the total protein of E. coli transformed with ΔpMA3S, and lane 7 is the total protein of E. coli transformed with ΔpMA5S. Lane 5 is a protein standard sample, a is phosphorylase B (106 kDa), b is bovine serum albumin (80 kDa), c is ovalbumin (49.5 kDa), and d is carbo. Nickic anhydrase (32.5 kDa), e denotes a soybean trypsin inhibitor (27.5 kDa) and f denotes lysozyme (18.5 kDa), respectively.
제9도에서 보듯이, pAI3UP와 pAI5UP로 형질전환된 대장균의 총단백질을 각각 나타내는 제10 및 11레인과 제12 및 15레인으로부터, 많은 양의 단백질 발현(화살표)이 있었음을 알 수 있었다.As shown in FIG. 9, there was a large amount of protein expression (arrow) from lanes 10 and 11 and 12 and 15, which represent the total protein of E. coli transformed with pAI3UP and pAI5UP, respectively.
[실시예 7]Example 7
재조합 인간 PTH의 발현 확인Expression of Recombinant Human PTH
전기 실시예 6에서 발현 벡터 pAI5UP와 pAI3UP로 형질전환된 대장균으로부터 대량으로 발현된 단백질이 인간 PTH인지의 여부를 확인하기 위하여, 안티-PTH 항체(anti-PTH antibody)를 이용하여 웨스턴 블럿(Western blot) 분석하였다. 즉, pAI5UP와 pAI3UP로 형질전환된 대장균 MC1061을 배양하여 총단백질을 SDS-PAGE로 분획한 다음, 총단백질을 니트로셀룰로스막(nitrocellulose membrane)에 옮겨, 안티-PTH 폴리클로날 항체(Biogenex, USA)를 사용하여 웨스턴 블럿(참조: Maniatis et al., Molecular Cloning 3, Cold Spring Harbor Laboratory, 1989) 분석하였다(참조: 제10도).In order to confirm whether the protein expressed in large quantities from E. coli transformed with the expression vectors pAI5UP and pAI3UP in the above Example 6 is human PTH, Western blot using an anti-PTH antibody ) Was analyzed. In other words, E. coli MC1061 transformed with pAI5UP and pAI3UP were cultured, and the total protein was fractionated by SDS-PAGE, and then the total protein was transferred to a nitrocellulose membrane and an anti-PTH polyclonal antibody (Biogenex, USA). Western blot (Maniatis et al., Molecular Cloning 3, Cold Spring Harbor Laboratory, 1989) was analyzed (Fig. 10).
제10도에서, 제1,2,3 및 4레인은 pAI3UP로 형질전환된 대장균의 총단백질, 제5,6,7 및 8레인은 pAI5UP로 형질전환된 대장균의 총단백질을 각각 나타내며; 제1 및 5레인은 L-arabinose로 유도하지 않고 하루동안 진탕배양한 대장균의 총단백질, 제2 및 6레인은 L-arabinose로 유도하기 직전의 대장균 총단백질, 제3 및 7레인은 L-arabinose로 유도한 다음 8시간 배양한 대장균의 총단백질, 제4 및 8레인은 L-arabinose로 유도한 다음 하루동안 배양한 대장균의 총단백질, 제9레인은 단백질 표준 시료물질로서 a는 포스포릴레이즈(106kDa), b는 소혈청 알부민(80kDa), c는 오발부민(49.5kDa), d는 카보닉 안하이드레이즈(32.5kDa), e는 소이빈 트립신 저해제(27.5kDa) 및 f는 라이소자임(18.5kDa)을 각각 나타낸다.In FIG. 10, lanes 1, 2, 3 and 4 represent the total protein of Escherichia coli transformed with pAI3UP, and lanes 5,6,7 and 8 represent the total protein of Escherichia coli transformed with pAI5UP, respectively; Lanes 1 and 5 did not induce L-arabinose, but the total protein of E. coli shaken for one day, and 2 and 6 were E. coli total protein immediately before L-arabinose, and 3 and 7 were L-arabinose. The total protein of E. coli cultured for 8 hours, and the 4th and 8th lanes were induced with L-arabinose and the total protein of E. coli cultured for 1 day, and the ninth lane was the protein standard sample. (106 kDa), b is bovine serum albumin (80 kDa), c is ovalbumin (49.5 kDa), d is carbonic anhydrase (32.5 kDa), e is soybean trypsin inhibitor (27.5 kDa), and f is lysozyme (18.5) kDa) is shown, respectively.
제10도에서 보듯이, L-arabinose로 유도한 pAI3UP로 형질전환된 대장균의 총단백질과 pAI5UP로 형질전환된 대장균의 총단백질을 나타내는 제3 및 4레인과 제7 및 8레인은 안티-PTH 항체와 특이적으로 반응하는 단일밴드를 나타내는 것으로 보아, 전기 형질전환된 대장균으로부터 발현된 단백질이 Ici A가 융합된 인간 PTH임을 알 수 있었다.As shown in FIG. 10, the 3rd and 4th lanes and the 7th and 8th lanes, which represent the total protein of E. coli transformed with pAI3UP induced by L-arabinose and the total protein of E. coli transformed with pAI5UP, are anti-PTH antibodies. It can be seen that the protein expressed from the E. coli transformed specifically with the human PTH fused with Ici A.
[실시예 8]Example 8
재조합 인간 PTH를 포함한 융합단백질의 단백질 봉입체 형성Protein inclusion body formation of fusion proteins including recombinant human PTH
L-arabinose 유도에 의하여 발현된 인간 PTH 대장균 세포내에서 봉입체(inclusion body)를 형성하는 것을 다음과 같이 확인하였다: 융합단백질의 발현을 유도한 다음, 진탕배양하고 원심분리하여 균체를 수득하였다. 전기에서 수닥한 균체에 리소자임(lysozyme, 100mg/g 세포)을 첨가하고 초음파 파쇄(sonication)하여 제조한 세포 파쇄액을 15,000rpm으로 원심분리하여, 봉입체를 수득하고 이를 여러번 세척하였다. 이어, 우레아를 이용하여 봉입체를 변성(denaturation)시킨 다음, 수득한 단백질을 재결합시켰다. 전기 봉입체 분리과정 중의 각 시료들을 SDS-PAGE하였다(참조: 제11도).Formation of inclusion bodies in human PTH Escherichia coli cells expressed by L-arabinose induction was confirmed as follows: Expression of the fusion protein was induced, followed by shaking culture and centrifugation to obtain cells. Lysozyme (100 mg / g cells) was added to the cells in the previous period and the cell lysate prepared by sonication was centrifuged at 15,000 rpm to obtain inclusion bodies and washed several times. Subsequently, the inclusion body was deaturated with urea and the obtained protein was recombined. Each sample was subjected to SDS-PAGE (see FIG. 11) during the electroinclusion separation process.
제11도에서, 제1레인은 L-arabinose로 유도한 후 20시간 배양한 pAI3UP로 형질전환된 대장균의 세포 파쇄액을 원심분리하여 수득한 상등액, 제2레인은 제1레인의 원심분리 후 수득한 침전물을 세척용 완충용액(0.5% Triton X-100, 1mM EDTA, 1mM DTT)으로 현탁시킨 다음, 다시 원심분리하여 수득한 상등액, 제3레인은 제2레인의 원심분리 후 수득한 침전물을 제2레인의 시료제조시와 동일하게 처리하고 원심분리하여 수득한 상등액, 제4레인은 제3레인의 원심분리 후 수득한 침전물에 8M 우레아 용액을 처리하고 원심분리하여 수득한 상등액, 제5 내지 8레인은 pAI5UP로 형질전환된 대장균을 이용하여 제1 내지 4레인과 동일하게 처리하여 수득한 상등액, 제9레인은 단백질 표준 시료물질로서 a는 오발부민(43kDa), b는 카보닉 안하이드레이즈(29kDa), c는 β-락트알부민(18.4kDa)및 d는 라이소자임(14.3kDa)을 각각 나타낸다. 제11도에서 보듯이, 봉입체의 단백질을 나타내는 제4 및 8레인에서 Ici A가 융합된 인간 PTH에 해당하는 단백질 띠(화살표)를 볼 수 있으므로, 전기 융합단백질은 대장균 내에서 상당량 발현되어, 모두 봉입체 형태로 분리됨을 알 수 있었다.In FIG. 11, the first lane is a supernatant obtained by centrifugation of the cell lysate of E. coli transformed with pAI3UP after induction with L-arabinose, and the second lane is obtained after centrifugation of the first lane. One precipitate was suspended in a wash buffer solution (0.5% Triton X-100, 1 mM EDTA, 1 mM DTT), and then the supernatant obtained by centrifugation again, and the third lane was the precipitate obtained after centrifugation of the second lane. The supernatant obtained by treating in the same manner as in the preparation of the two lanes and centrifuging, the fourth lane is the supernatant obtained by treating 8M urea solution and centrifuging the precipitate obtained after centrifugation of the third lane, and 5 to 8. Lane is a supernatant obtained by the same treatment as in the first to fourth lanes using E. coli transformed with pAI5UP, the ninth lane is a protein standard sample material, a is ovalbumin (43kDa), b is carbonic anhydrase ( 29 kDa), c is β-lactalbumin (18.4 kDa) and d is It shows the isozyme (14.3kDa) respectively. As shown in FIG. 11, since the protein bands (arrows) corresponding to Ici A-fused human PTH can be seen in the 4th and 8th lanes of the inclusion body protein, the electric fusion protein is expressed in a large amount in E. coli. It can be seen that it is separated into inclusion bodies.
[실시예 9]Example 9
유로키나제를 이용한 재조합 인간 PTH의 분리Isolation of Recombinant Human PTH Using Eurokinase
전기 실시예 8과 같이 분리한 봉입체에 존재하는 Ici A/PTH 융합단백질의 양을 측정한 다음, 트리스 완충용액(0.1M NaCl 및 20μM DTT를 함유한 20mM 트리스, pH 7.8)으로 단백질 농도가 1mg/m1이 되도록 조정하였다.The amount of Ici A / PTH fusion protein present in the inclusion bodies isolated as in Example 8 was measured, and then the protein concentration was 1 mg / t with Tris buffer solution (20 mM Tris, pH 7.8 containing 0.1 M NaCl and 20 μM DTT). It adjusted to m1.
그런 다음, 단백질 분해효소인 유로키나제를 넣고 25℃에서 반응시켜, 융합단백질로부터 인간 PTH를 분리하였다(참조: 제12(A)도). 제12(A)도에서, 제1레인은 유로키나제 처리 후의 Ici A/PTH 융합단백질을 전기영동한 결과, 제3레인은 표준 단백질의 분자량 마커를 각각 나타낸다. 제12(A)도에서 보듯이, 유로키나제에 의해 Ici A/PTH 융합단백질로부터 PTH가 분리되었음을 알 수 있었다.Then, urokinase, a protease was added thereto, and reacted at 25 ° C to separate human PTH from the fusion protein (see also Figure 12 (A)). In FIG. 12 (A), the first lane electrophoresed the Ici A / PTH fusion protein after urokinase treatment, and the third lane shows the molecular weight marker of the standard protein, respectively. As shown in FIG. 12 (A), it was found that PTH was separated from Ici A / PTH fusion protein by urokinase.
제12(b)도는 분리된 PTH 단백질을 정제하여 SDS-PAGE하고 실버 염색(silver staining)으로 분석한 결과인데, 제1레인은 표준 단백질의 분자량 마커, 제2레인은 5μg의 정제된 PTH, 제3레인은 12μg의 정제된 PTH를 각각 나타낸다. 제12(B)도에서 보듯이, 인간 재조합 PTH는 순수하게 정제되었음을 알 수 있었다.Figure 12 (b) is the result of the purification of the isolated PTH protein by SDS-PAGE and silver staining (silver staining), the first lane is the molecular weight marker of the standard protein, the second lane is 5μg purified PTH, Three lanes each represent 12 μg of purified PTH. As shown in FIG. 12 (B), it was found that the human recombinant PTH was purified purely.
[실시예 10]Example 10
인간 재조합 PTH의 활성측정Activity of Human Recombinant PTH
UMR106 세포주(rat osteoblast-like osteosarcoma cell line)는 골생성세포(osteoblast)의 특성을 연구하는 재료로서 많이 이용되고 있는데, 골생성세포의 특징은 알칼리성 포스파타제(alkaline phosphatase)의 활성이 높으며, 타잎 1 콜라겐을 생산하는 것으로 알려져 있다(참조: Meika A. Fang et al., Endocrinology, 131(5):2113-2119(1992); Cheryl O. Quinn et al., The Journal of Biological Chemistry, 265(36):22342-22347(1990)).UMR106 cell line (rat osteoblast-like osteosarcoma cell line) is widely used as a material for studying the characteristics of osteoblasts (osteoblasts), characterized in that the activity of the alkaline phosphatase (alkaline phosphatase), type 1 collagen Meika A. Fang et al., Endocrinology, 131 (5): 2113-2119 (1992); Cheryl O. Quinn et al., The Journal of Biological Chemistry, 265 (36): 22342-22347 (1990).
따라서, 실시예 9에서 정제한 재조합 PTH(rhPTH-(1-84))의 시험관내 생물학적 활성은 UMR106 세포주(ATCC CRL 1661)를 이요하여 PTH의 수용체에 대한 결합력 시험과 세포내 cAMP 생성촉진 시험으로 측정하였다. 대조군으로는 합성 PTH(shPTH-(1-84), Sigma Chemical Co., U.S.A.)를 사용하였으며, 아미노 말단과 카르복시 말단을 가진 완전한 PTH만이 검출되는 Allegro Intact PTH RIA kit(Nichols Institute, San Juan Capistrano, U.S.A.)를 사용하여 PTH를 정량하였다(참조: Samuel R. Nussbaum et al., Clinical Chemistry, 33(8):1364-1367(987)). 아울러, UMR106 세포주는 0.2% 소디움바이카보네이트 및 10% FB S(fetal bovine serum, 56℃에서 30분간 열처리한것)를 함유한 DMEM(Dulbecco's Modified Eagle Medium)에서, 37℃, 5% CO2의 조건으로 배야하였다(참조: Ronald J. Midura et al., The Journal of Biological Chemistry, 269(18):13200-13206(1994)).Therefore, the in vitro biological activity of the recombinant PTH (rhPTH- (1-84)) purified in Example 9 was tested by the UMR106 cell line (ATCC CRL 1661) to test the binding ability of PTH to the receptor and the intracellular cAMP production promotion test. Measured. As a control, synthetic PTH (shPTH- (1-84), Sigma Chemical Co., USA) was used, and Allegro Intact PTH RIA kit (Nichols Institute, San Juan Capistrano, USA) was used to quantify PTH (Samuel R. Nussbaum et al., Clinical Chemistry, 33 (8): 1364-1367 (987)). In addition, the UMR106 cell line was treated with DMEM (Dulbecco's Modified Eagle Medium) containing 0.2% sodium bicarbonate and 10% FB S (fetal bovine serum, heat-treated at 56 ° C. for 30 minutes) at 37 ° C. and 5% CO 2 . Ronald J. Midura et al., The Journal of Biological Chemistry, 269 (18): 13200-13206 (1994).
[실시예 10-1]Example 10-1
PTH의 수용체에 대한 결합력 시험Binding test for receptors of PTH
PTH의 수용체에 대한 결합력 시험은 다음과 같이 진행하였다9참조: Chohei Shigeno et al., The Journal of Biological Chemistry, 263(8): 3864-3871(1988)): 24공(well) 배양기에 공당 105개의 UMR106 세포주를 접종하여 4 내지 8일간 배양하였는데, 이때 배지는 이틀마다 한번씩, 시험하기 3일전부터는 매일 교체하였다. 한편, 카르복실 말단의 티로신 잔기에 방사선 동위원소125I이 표지된[Nle8,18, Tyr34]-소(bovine)의 PTH-(1-34) NH2(bPTH-(1-34))(참조: Gino V. Segre et al., The Journal of Biological Chemistry, 254(15):6980-6986(1979))를 제조하기 위하여, 반응 촉매제로서 클로라민 T(chloramine T, Sigma Chemical Co., U.S.A.)를 사용하여 bPTH-(1-34)을 Na125I로 요오드화 반응(iodination)시킨다음, C18-팩컬럼에 주입하고 반응하지 않는125I을 제거하였다. 그런 다음, 요오드화된 bPTH를 50% ACN(acetonitrile)-0.1% TFA(trifluoroacetic acid)로 용출시키고, ACN을 제거하였다.Adhesion testing of PTH to receptors proceeded as follows: Chohei Shigeno et al., The Journal of Biological Chemistry, 263 (8): 3864-3871 (1988)): Five UMR106 cell lines were inoculated and incubated for 4-8 days, with the medium replaced once every two days, daily from three days prior to testing. On the other hand, PTH- (1-34) NH 2 (bPTH- (1-34)) of [Nle 8,18 , Tyr 34 ] -bovine labeled with radioisotope 125 I at the tyrosine residue at the carboxyl terminus. To prepare (Gino V. Segre et al., The Journal of Biological Chemistry, 254 (15): 6980-6986 (1979)), chloramine T (Sigma Chemical Co., USA) as a reaction catalyst BPTH- (1-34) was iodized with Na 125 I, then injected into a C 18 -pack column to remove 125 I unreacted. Iodide bPTH was then eluted with 50% ACN (acetonitrile) -0.1% trifluoroacetic acid (TFA) and ACN was removed.
상기에서 제조한125I이 표지된 [Nle8.18, Tyr34]-소의 PTH-(1-34) NH2(bPTH-(1-34))를 리간드로 사용하여, 이 리간드가 수용체에 결합하는 것을 rhPTH-(1-84) 및 shPTH-(1-84)가 경쟁적으로 제해하는 정도를 비교함으로써, PTH의 수용체에 대한 결합력을 측정하였다. 리간드와 전기 PTH들을 결합 완충용액(100mM Nacl, 5mM KCl, 2mM CaCl2, 5% 말의 혈청(horse serum) 및 0.5% FBS을 함유한 50mM 트리스, pH 7.7)에 각각 희석하였다.Using the above-prepared 125 I-labeled [Nle 8.18 , Tyr 34 ] -bovine PTH- (1-34) NH 2 (bPTH- (1-34)) as a ligand, the ligand binds to the receptor. By comparing the degree of competitive inhibition of rhPTH- (1-84) and shPTH- (1-84), binding of PTH to the receptor was measured. Ligand and electrical PTH were diluted in binding buffer (100 mM Nacl, 5 mM KCl, 2 mM CaCl 2 , 50 mM Tris, 5% horse serum and 0.5% FBS, pH 7.7), respectively.
한편, 상기에서 배양한 24공 배양기를 얼음위에서 냉각시키고 1ml의 결합완충용액으로 2회 세척한 후, 30000 내지 50000cpm의 리간드와 농도를 달리한 경쟁 PTH를 최종부피가 0.3ml되게 첨가하였다. 각 호르몬의 희석물은 3개의 공에 동시 처리하였으며, 15℃에서 4시간동안 흡착시켰다. 반응이 끝난 후, 0.5ml의 결합완충용액으로 4회 세척하여, 결합하지 않은 방사능을 제거하였다. 세포에 결합한 방사능을 0.5ml의 0.5M NaOH를 가하여 실온에서 16 내지 18시간 추출하고, 0.5ml의 결합완충용액으로 재차 세척한 세척물을 합쳐 감가 계수기(γ-counter)로 방사능을 측정하였다.Meanwhile, the 24-hole incubator cultured in the above was cooled on ice and washed twice with 1 ml of binding buffer solution, and the final volume was added to the final volume of 0.3 ml of 30000 to 50000 cpm ligand and a different concentration. Dilutions of each hormone were simultaneously treated in three balls and adsorbed at 15 ° C. for 4 hours. After the reaction was completed, washed four times with 0.5ml binding buffer solution, to remove the unbound radioactivity. The radioactivity bound to the cells was extracted by adding 0.5 ml of 0.5 M NaOH at room temperature for 16 to 18 hours, and the washings washed again with 0.5 ml of the binding buffer solution were combined and the radioactivity was measured by a decrement counter (γ-counter).
전기에서 측정한 결합된 방사능 측정치에서 1mM의 각 경쟁 호르몬을 사용하여 측정한 비특이적 결합의 측정치를 감해 주어, 특이적 결합의 측정치를 결정하였다. 측정결과는 최대 특이적 결합의 백분율을 표시하였으며, 곡선(curve)의 최적화 및 각 경쟁 호르몬들의 결합력을 비교하는 중요한 지표가 되는 IC50(50% inhibitory concentration, 리간드의 결합을 50% 저해시키는데에 필요한 호르몬의 농도)의 값은 Biosoft사의 fig. P program을 사용하여 결정하였다. shPTH-(1-84)와 rhPTH-(1-84)의 IC50값은 각각 18.6±1.5 및 17.3±3.1nM(평균치±표준편차)로 나타났으며, 그 중 하나의 실험결과를 제13(A)도에 나타내었다. 제13(A)도에서 보듯이, 재조합 PTH는 합성 PTH와 대등한 수용체 결합력을 가지고 있음을 알 수 있었다.The measurement of specific binding was determined by subtracting the measurement of nonspecific binding measured using 1 mM of each competing hormone from the measured radioactivity measured earlier. The measurement results indicate the percentage of maximum specific binding, which is required for IC 50 (50% inhibitory concentration, 50% inhibition of ligand binding), which is an important indicator of the optimization of the curve and the comparability of each competing hormone. Hormone concentration). It was determined using the P program. IC 50 values of shPTH- (1-84) and rhPTH- (1-84) were 18.6 ± 1.5 and 17.3 ± 3.1nM (mean ± standard deviation), respectively. A) is shown in the figure. As shown in FIG. 13 (A), the recombinant PTH was found to have the same receptor binding capacity as the synthetic PTH.
[실시예 10-2]Example 10-2
세포내 cAMP생성 촉진시험Intracellular cAMP production promotion test
세포내 cAMP생성 촉진시험은 다음과 같이 진행하였다(참조: Thomas J. Gardella et al., The Journal of Biological Chemistry, 265(26): 15854-15859(1990)). 실시예 10-1에서 4내지 8일간 24공 배양기에서 배양한 UMR106세포를 얼음위에서 15분간 냉각시킨 후, 0.25ml의 cAMP 완충용액(2mM 3-isobuty1-methylxanthine, 1mg/ml bovine serum albumin, 35mM HEPES를 함유하는 DMEM, ph 7.4)으로 1회 세척하였다. 그런 다음, 0.1ml의 cAMP완충용액을 가한 후, 0.1ml의 각각 다른 농도의 호르몬이 희석된 cAMP 완충용액을 첨가하여 37℃에서 20분간 반응시키고, 완충액을 제거하였다.Intracellular cAMP production facilitation was conducted as follows (see Thomas J. Gardella et al., The Journal of Biological Chemistry, 265 (26): 15854-15859 (1990)). After cooling UMR106 cells cultured in a 24-hole incubator for 4 to 8 days in Example 10-1 for 15 minutes on ice, 0.25 ml of cAMP buffer (2 mM 3-isobuty1-methylxanthine, 1 mg / ml bovine serum albumin, 35 mM HEPES) Washed once with DMEM, ph 7.4). Then, 0.1 ml of cAMP buffer solution was added, and then 0.1 ml of different concentrations of hormones diluted with cAMP buffer were added to react for 20 minutes at 37 ° C., and the buffer solution was removed.
이어, -70℃에서의 동결 및 실온에서의 해동을 각각 20분간 3회 반복하여 세포를 파괴하고, 1ml의 50mM HCl을 각 공에 가한 다음, -20℃에서 16 내지 18시간 동안 세포내 cAMP를 추출하고, 그 추출물에 있는 cAMP을 cAMP RIA kit(New England Nuclear, Du Pont, U.S.A.))를 사용하여 정량하였다. 곡선(curve)의 최적화 및 EC50(세포내 cAMP의 생성을 50% 촉진시키는데 필요한 호르몬의 농도)의 값은 Biosoft사의 fig.P program을 사용하여 결정하였다. shPTH-(1-84) 및 rhPTH-(1-84)의 EC50수치는 각각 1.9±0.1와 1.3±0.2nM(평균치±표준편차)로 나타났으며, 그 중 하나의 실험결과를 제13(B)도에 나타내었다. 제13(B)도에서 보듯이, 재조합 PTH는 합성 PTH와 대등한 아데닐레이트 사이클레이즈의 활성을 가지고 있음을 알 수 있었다.Subsequently, cells were destroyed by freezing at −70 ° C. and thawing at room temperature three times for 20 min each, adding 1 ml of 50 mM HCl to each ball, and then intracellular cAMP at −20 ° C. for 16 to 18 hours. The cAMP in the extract was quantified using the cAMP RIA kit (New England Nuclear, Du Pont, USA). Optimization of the curve and the value of EC 50 (hormonal concentration required to promote 50% production of intracellular cAMP) was determined using the Fig. P program from Biosoft. EC 50 values of shPTH- (1-84) and rhPTH- (1-84) were 1.9 ± 0.1 and 1.3 ± 0.2nM (mean ± standard deviation), respectively. B) is shown in FIG. As shown in FIG. 13 (B), the recombinant PTH was found to have the activity of adenylate cyclase comparable to the synthetic PTH.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 융합 파트너로 이용되는 Ici A 유전자 단편을 함유한 L-arabinose 유도성 벡터에, 유로키나제 절단부위의 뉴클레오티드 서열을 포함한 PTH의 유전자를 삽입하여 제조한 재조합 발현벡터, 그로부터 형질전환된 재조합 미생물 및 전기 미생물을 L-arabinose가 첨가된 배지에서 배양함으로써 인간 부갑상선 호르몬을 대량으로 제조하는 방법을 제공하는 것이다. 본 발명에서 제조된 재조합 인간 PTH 천연형 인간 PTH의 활성을 가지고 있음을 확인하였다. 따라서, 본 발명의 재조합 발현벡터로 형질전환된 미생물을 배양하여 L-arabinose로 발현을 유도함으로써, 천연형 인간 PTH가 지닌 활성을 그대로 유지하고 있는 재조합 인간 PTH를 정확한 유도조절을 통하여 고수율로 제조할 수 있다.As described and demonstrated in detail above, the present invention provides a recombinant expression vector prepared by inserting a PTH gene including a nucleotide sequence of a urokinase cleavage site into an L-arabinose inducible vector containing an Ici A gene fragment used as a fusion partner. The present invention provides a method for producing human parathyroid hormone in large quantities by culturing recombinant microorganisms and electric microorganisms transformed therefrom in a medium to which L-arabinose is added. It was confirmed that the recombinant human PTH prepared in the present invention has the activity of native human PTH. Therefore, by inducing the expression of L-arabinose by culturing the microorganism transformed with the recombinant expression vector of the present invention, the recombinant human PTH that maintains the activity of the native human PTH as it is produced in high yield through accurate induction control can do.
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