KR940005594B1 - Method of refinning o-growth hormones - Google Patents
Method of refinning o-growth hormones Download PDFInfo
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Abstract
Description
제1도는 형질전환된 대장균으로 부터 양 성장 호르몬의 분리 정제개요를 나타낸 것이다.Figure 1 shows the separation and purification of sheep growth hormone from the transformed E. coli.
제2도는 세포내 불용성 단백질 분리 및 용해 결과를 전기영동으로 나타낸 것이다.Figure 2 shows the results of electrophoresis of intracellular insoluble protein separation and lysis.
제3도는 DEAE 음이온교환수지 크로마토그래피 결과를 나타낸 것이다.Figure 3 shows the DEAE anion exchange resin chromatography results.
제4도는 S-200 겔 투과 크로마토그래피 결과를 나타낸 것이다.4 shows the results of S-200 gel permeation chromatography.
제5도는 재조합 양 성장 호르몬의 생물활성도를 나타낸 것이다.5 shows the bioactivity of recombinant sheep growth hormone.
본 발명은 양 성장 호르몬(Ovine Growth Hormone : OGH)의 정제 방법에 관한 것으로, 보다 상세하게는 유전공학적인 방법에 의해 제조합된 발현벡터로 형질전환된 대장균 세포로부터 대량 발현된 순도가 낮은 양 성장 호르몬을 고순도로 양 성장 호르몬으로 정제하는 방법에 관한 것이다.The present invention relates to a method for purifying Ovine Growth Hormone (OGH), and more particularly, low-purity sheep growth expressed in large quantities from E. coli cells transformed with expression vectors prepared by genetic engineering methods. The present invention relates to a method for purifying hormones with high growth hormone in high purity.
종래에는 축산업분야에서 주로 고단백질 사료나 스테로이드 계통 호르몬을 사료에 섞어 사용함으로써 사료 효율 및 가축의 체중 증가율을 높여 왔다. 그러나, 고단백질 사료의 경우에는 그 원천이 한정되어 있으며, 또한, 스테로이드 물질은 사용 후 오랜기간 동안 동물에 잔류하여 인간이 섭취할 경우 악 영향을 받는다는 것이 미국을 비롯한 선진 각국에서 확인되어 점점 그 사용이 금지되고 있다. 이에 반해 최근에 대두되고 있는 동물 성장 호르몬은 섭취하더라도 체내에 잔류하지 않고 종 특이성(species-specificity)을 나타내어 사람에게 영향을 주지 않으므로 사료 효율을 높일 수 있는 이상적인 물질로서, 특히 성장 호르몬을 양에게 투여하면 체중이 증가할 뿐만 아니라 사료효율 및 양털 성장 촉진을 일으키는 것으로 알려지고 있다.Conventionally, in the livestock industry, high-protein feed or steroid-based hormones are mixed with feed to increase feed efficiency and weight gain of livestock. However, in the case of high protein feed, its source is limited, and it has been confirmed in developed countries including the United States that steroid substances remain in animals for a long time after use and are adversely affected by human ingestion. This is forbidden. On the other hand, animal growth hormone, which is recently emerging, does not remain in the body even when ingested and shows species-specificity, which does not affect humans. It is known to increase body weight as well as promote feed efficiency and fleece growth.
따라서, 본 발명의 목적은 유전공학적인 방법을 이용하여 경제적이면서 대량으로 양 성장 호르몬을 대장균으로부터 분리·정제하여 양(羊) 축산업에 사료성분으로 이용하는 것이다.Accordingly, an object of the present invention is to economically and massively separate and purify sheep growth hormone from E. coli using genetic engineering methods, and to use them as feed ingredients in the livestock industry.
동물 성장 호르몬은 조직의 성장을 촉진시키고 단백질, 지방 및 탄수화물 대사를 조절하는 단백질 중의 하나로서, 단일가닥 구형 단백질로서 분자량은 22K 내지 24K 달톤(daltons)이며 190 내지 120개의 아미노산으로 구성되어 있다. (Barrington, E. J., Comparative Biochemistry of Growth Hormone, Prolactin, and Glycoprotein Hormones : Hormones : Hormone : Hormone and Evolution, Academin Press, N. Y., Vol.2(1979). 이러한 호르몬들은 뇌하수체 전엽에서 분리되며, 일반적으로 종 특이성을 가지고, 종에 따라서 그 등전점(pI), N말단과 C말단의 아미노산 및 전체 아미노산 구성 비율이 약간씩 다르다. 사람과 소, 양 및 쥐 성장 호르몬의 아미노산 서열은 이미 잘 알려져 있으며 (Dayhoff, M.D.(1976), Atlas of Protein Sequences and Structure, National Biomedical Research Foundation, Vol 5 : Supple.2), 이들 동물 성장 호르몬은 세포내에서 아미노 말단에 "시그날(signal)"시퀀스를 갖고 있는 전구물질로서 합성되어 ER(endoplasmic reticulum)막을 거쳐 시그날 시퀀스가 절단되어 성숙형 성장 호르몬으로 만들어진다(Davies et al., Nature, 283, 433-438(1980)).Animal growth hormone is one of proteins that promotes tissue growth and regulates protein, fat and carbohydrate metabolism. It is a single-stranded spherical protein having a molecular weight of 22K to 24K daltons and composed of 190 to 120 amino acids. (Barrington, EJ, Comparative Biochemistry of Growth Hormone, Prolactin, and Glycoprotein Hormones: Hormones: Hormone: Hormone and Evolution, Academin Press, NY, Vol. 2 (1979) .These hormones are isolated from the anterior pituitary gland and are generally species specific. And the ratio of the isoelectric point (pI), amino acids at the N- and C-terminal to total amino acid composition differs slightly from species to species, and the amino acid sequences of human, bovine, sheep and rat growth hormones are well known (Dayhoff, MD). (1976), Atlas of Protein Sequences and Structure, National Biomedical Research Foundation, Vol 5: Supple. 2), these animal growth hormones are synthesized as precursors with "signal" sequences at the amino terminus in cells. The signal sequence is cleaved through the endoplasmic reticulum (ER) membrane to produce mature growth hormone (Davies et al., Nature, 283, 433-438 (1980)).
성장 호르몬은 어린 동물들의 성장촉진을 위해 요구되기 때문에 특히 축산업에 유용하게 이용될 수 있다. 이들은 주로 아미노산들의 세포내 전달을 촉진시키고 전령 RNA의 해독을 촉진 시킴으로써 궁극적으로 동물들의 성장을 촉진시킨다. 또한 이들은 성장에 중요한 요인인 세포의 분열작용을 촉진시킨다. 따라서 성장호르몬을 투여 받은 동물은 조직단백질이 증가되어 사료량의 증가없이 부가적인 체중의 증가로 인하여 사료의 효율을 증대시킬 수 있다.Growth hormone is particularly useful in the livestock industry because it is required for the growth of young animals. They mainly promote the growth of animals by primarily promoting the intracellular delivery of amino acids and the translation of messenger RNA. They also promote cell division, an important factor for growth. Therefore, animals receiving growth hormone can increase the efficiency of feed due to the increase in tissue protein and additional weight gain without increasing the amount of feed.
한편, 축산업에서 이러한 호르몬을 이용하려면 많은 양이 필요하기 때문에 뇌하수체에서 성장 호르몬을 분리하여 사용하기는 어려웠다. 지금까지 인간, 돼지, 소 및 쥐 등의 동물 성장 호르몬들이 클로닝되어 대장균 세포내에서 발현되었다(Miller et. al., JBC,255, 7521-7524(1980) ; Martial et al., Science205,602-607(1979) ; Seeburg et al., Nature,270, 486-494(1977)).On the other hand, it is difficult to separate the growth hormone from the pituitary gland because it requires a large amount to use these hormones in the livestock industry. To date, animal growth hormones of humans, pigs, cattle and rats have been cloned and expressed in E. coli cells (Miller et. Al., JBC, 255 , 7521-7524 (1980); Martial et al., Science 205, 602). -607 (1979); Seeburg et al., Nature, 270 , 486-494 (1977).
본 발명자들은 본 출원인이 선 출원한 한국 특허출원 제91-17583호에 개시되어 있는 바와 같이, 양 성장호르몬의 유전자를 합성하여 재조합 발현벡터를 작제한 후 대장균(E. coli, W3110)에 형질전환시킨 미생물(E. coli, W3110(ptrp-OST), 기탁번호 : KCCM-10006, 기탁기관 : 한국미생물보존센터(KCCM), 기탁일 : 1991년 6월 26일)에서 발현된 순도가 낮은 양 성장 호르몬을 정제하는데 있어서, 초음파를 이용한 세포파괴 과정 및 불용해 단백질제거 과정, 우레아를 이용한 용해과정, 음이온 교환 수지 크로마토그래피 과정, 겔 투과 크로마토그래피 과정을 거침으로써 경제적으로 고순도의 양 성장 호르몬을 얻는 방법을 알아내고 본 발명을 완성하게 되었다.The inventors of the present invention, as disclosed in Korean Patent Application No. 91-17583 filed by the applicant, synthesized the genes of both growth hormones and constructed a recombinant expression vector, and then transformed into E. coli (W3110). Low-purity sheep expressed in E. coli, W3110 (ptrp-OST), Deposit No .: KCCM-10006, Deposit Organization: Korea Microorganism Conservation Center (KCCM), Deposit Date: June 26, 1991) In the purification of hormones, a method of obtaining high-purity sheep growth hormone economically by performing cell destruction process using ultrasonic waves, insoluble protein removal process, urea dissolution process, anion exchange resin chromatography process, and gel permeation chromatography process. The present invention was completed.
본 발명을 상세하게 설명하면 다음과 같다.The present invention will be described in detail as follows.
양 성장 호르몬의 유전자를 포함하는 형질전환된 대장균 세포(KCCM-10006)를 앰피실린(ampicillin)과 트립토판(tryptophan)이 함유된 M9 배지에 옮겨 배양하고, 흡광도 OD600이 0.7에 도달하면 인돌 아크릴산(indoleacrylic acid)을 첨가한 후, 더 배양한 다음 원심분리기를 이용하여 대장균 세포를 침전시키고 침전된 세포들을 트리스 완충용액에 현탁시킨다. 초음파 세포파쇄기로 처리하여 세포를 파쇄한 다음, 원심 분리기로 원심분리하여 침전물을 취한다. 침전물을 세척한 후 트리스 완충용액을 넣고 상온에서 교반시킨다. 이 용액을 원심분리기로 원심분리하여 상층액을 취한 다음 탄산 암모니아 완충용액을 가하여 교반시킨다. 그런 다음 원심분리하여 상층액을 탄산 암모니아 완충용액으로 평형시킨 음이온 교환수지 칼럼에 통과시켜 양 성장 호르몬을 부착시키고, 컬럼내 유리된 상태로 남아있는 물질은 탄산 암모니아 완충용액으로 세척한다. 수지에 부착된 단백질을 0-0.5M NaCl 직선 농도 구배를 주어 분리하면, 양 성장 호르몬은 0.1M-0.2M NaCl 사이에서 용출된다. 용출된 각 부분을 전기 영동한 후 양 성장 호르몬 부분을 모은 후 한외여과막(ultrafiltration membrane)을 이용하여 농축하거나 혹은 황산 암모늄을 첨가하여, 30% 포화농도로 만들어 양 성장 호르몬을 침전시킨 후 탄산 암모니아 용액에 용해하여 농축시킨 다음, S-200수지 컬럼에 통과시켜 용출된 부분 중 양 성장 호르몬 순도가 95% 이상인 부분만을 취한다.Transfected Escherichia coli cells (KCCM-10006) containing genes of both growth hormones were transferred to M9 medium containing ampicillin and tryptophan, and indoleacrylic when the absorbance OD600 reached 0.7. acid), followed by further incubation, and then the E. coli cells are precipitated using a centrifuge and the precipitated cells are suspended in Tris buffer. The cells are disrupted by treatment with an ultrasonic cell crusher and then centrifuged with a centrifuge to obtain a precipitate. After washing the precipitate, the Tris buffer solution was added and stirred at room temperature. The solution was centrifuged with a centrifuge to obtain the supernatant and then stirred by addition of ammonia carbonate buffer. After centrifugation, the supernatant is passed through an anion exchange resin column equilibrated with ammonia carbonate buffer to attach both growth hormones, and the remaining material in the column is washed with ammonia carbonate buffer. Proteins attached to the resin are separated by a 0-0.5M NaCl linear concentration gradient, and both growth hormones are eluted between 0.1M-0.2M NaCl. Each eluted part was electrophoresed, and then the sheep growth hormone was collected and concentrated using an ultrafiltration membrane, or ammonium sulfate was added to make 30% saturation to precipitate the sheep growth hormone. After dissolving and concentrating, the resultant is passed through an S-200 resin column, and only the portion of the eluted portion having a growth hormone purity of 95% or more is taken.
이하 본 발명을 실시예에 의거 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail with reference to Examples.
하기 실시예들은 본 발명의 구체적인 설명를 위한 것으로 본 발명의 사상을 벗어나지 않은 한, 본 발명의 범위를 제한하지 않는다.The following examples are provided for the detailed description of the present invention and do not limit the scope of the present invention without departing from the spirit of the present invention.
[실시예 1]Example 1
박테리아 세포 배양과정Bacterial Cell Culture
양 성장 호르몬 유전자가 클로닝된 대장균을 20㎍/ml 앰피실린과 50㎍/ml 트립토판이 함유된 20ml 루리아배지(Luria broth)에서 30℃에서 12시간 진탕 배양한 다음 500ml M9배지에 옮겨 3-4시간 배양하여 OD600이 0.6에 도달하면, 에탄올에 용해시킨 0.2M 인돌 아크린산(indoleacrylic acid)을 0.75ml 첨가한 다음 12시간 진탕 배양한 후 세포 배양액을 Beckman J-6B 원심분리기(rotor JS 4.2)로 3500rpm에서 30분간 원심분리하여 박테리아를 침전시켰다.Escherichia coli cloned with both growth hormone genes were shaken in a 20 ml Luria broth containing 20 µg / ml ampicillin and 50 µg / ml tryptophan for 12 hours at 30 ° C, and then transferred to 500 ml M9 medium for 3-4 hours. When the OD600 reached 0.6 by incubation, 0.75 ml of 0.2M indole acrylic acid dissolved in ethanol was added, followed by shaking culture for 12 hours, and then the cell culture was transferred to a Beckman J-6B centrifuge (rotor JS 4.2). The bacteria were precipitated by centrifugation at 3500 rpm for 30 minutes.
[실시예 2]Example 2
세포파괴 및 불용해 단백질 분리과정Cell destruction and insoluble protein separation process
침전된 발현된 대장균 세포 4g을 4℃의 30ml 50mM 트리스 완충용액(pH 8.0), 5mM EDTA, 5mM 베타머캡토에탄올에 현탁시킨 후, 얼음조에서 20분간 초음파 세포파쇄기(Heatsystem-Ultrasonic사, 미국)로 처리하여 세포를 파괴하였다. 이 파쇄액에 상기 세포파쇄 완충용액을 150ml 첨가한 후 1분간 교반시킨 다음, Beckman J2-21 원심분리기 (rotor JA-14)로 5500rpm에서 20분간 원심분리하여 상층액을 버리고 침전물은 200ml 1M NaCl, 5mM EDTA. 5mM 베타머캡토에탄올을 50mM 트리스 완충용액을 가하였다. 10분간 교반시킨 후 원심분리하여 상층액은 버리고 침전물을 200㎖ 4M 우레아, 2mM EDTA, 5mM 베타머캡토에탄올이 포함 된 50mM 트리스 완충용액(pH 8.0)에 현탁시켰다. 10분간 상온에서 교반시킨 후 Beck man J2-21 원심분리기(rotor JA14)로 800rpm에서 20분간 원심분리하여 그 침전물을 이용하였다.4 g of the expressed E. coli cells precipitated were suspended in 30 ml 50 mM Tris buffer (pH 8.0), 5 mM EDTA, 5 mM betamercaptoethanol at 4 ° C., and then ultrasonic cell crusher (Heatsystem-Ultrasonic, USA) for 20 minutes in an ice bath. Treated to destroy the cells. 150ml of the cell disruption buffer solution was added to the lysate, followed by stirring for 1 minute, followed by centrifugation at 5500 rpm for 20 minutes with a Beckman J2-21 centrifuge (rotor JA-14). The supernatant was discarded and the precipitate was 200ml 1M NaCl, 5 mM EDTA. 5 mM betamercaptoethanol was added to 50 mM Tris buffer. After stirring for 10 minutes, the supernatant was discarded by centrifugation, and the precipitate was suspended in 50 mM Tris buffer solution (pH 8.0) containing 200 ml 4M urea, 2 mM EDTA, and 5 mM betamercaptoethanol. After stirring at room temperature for 10 minutes, the precipitate was used by centrifugation at 800 rpm for 20 minutes with a Beck man J2-21 centrifuge (rotor JA14).
[실시예 3]Example 3
불용해 양 성장호르몬의 용해 과정Dissolution process of insoluble sheep growth hormone
상기 실시예에서 얻은 양 성장 호르몬을 포함하는 대장균 세포내의 불용해 단백질 침전물에 20ml의 8M우레아, 5mM EDTA, 5mM DTT(dithiothreital), 10mM 트리스 완충용액(pH 8.0)을 넣고 2시간 동안 상온에서 교반시켰다. 이 용액을 원심분리기(Beckman J2-21, rotor JA21, 미국)로 15000rpm에서 20분간 원심분리하여 불용해 물질을 제거하고 용해된 양 성장 호르몬이 있는 상층액을 취하였다. 상층액에 5mM 탄산암모니아 완충용액(pH 9.0)을 가하여 1M 우레아 농도가 되도록 한 후 1시간 동안 교반시킨 다음 다시 원심분리하여 침전물을 제거하였다.20 ml of 8M urea, 5 mM EDTA, 5 mM dithiothreital, and 10 mM Tris buffer solution (pH 8.0) were added to an insoluble protein precipitate in E. coli cells containing sheep growth hormone obtained in the above example, and stirred at room temperature for 2 hours. . The solution was centrifuged in a centrifuge (Beckman J2-21, rotor JA21, USA) for 20 minutes at 15000 rpm to remove insoluble material and supernatant with dissolved sheep growth hormone. 5mM ammonia carbonate buffer solution (pH 9.0) was added to the supernatant to a concentration of 1M urea, stirred for 1 hour, and then centrifuged to remove the precipitate.
[실시예 4]Example 4
음이온 교환 수지 컬럼 크로마토그래피 과정Anion Exchange Resin Column Chromatography Process
상기 실시예 3에서 얻어진 양 성장 호르몬을 포함하는 상층액을 1M 우레아 5mM 탄산암모니아 완충용액(pH 9.6)으로 평형된 DE52 이온교환수지(Whatman사, 미국)컬럼(2.5cm×10cm)에 10ml/cm2/hour 속도로 통과시켜 양 성장 호르몬을 부착시킨 다음, 컬럼내 유리된 상태로 남아 있는 물질들과 우레아를 5mM 탄산암모니아 용액(pH 9.6)으로 세척하였다. 수지에 부착된 단백질을 300ml의 5mM 탄산암모니아 용액(pH 9.6)과 300ml의 0.5M NaCl 5mM 탄산암모니아 용액을 염직선 농도 구배를 만들어 용출시켰으며, 이때 양 성장 호르몬은 0.1M-0.2M NaCl 농도에서 용출되었다.10 ml / cm in a DE52 ion exchange resin (Whatman, USA) column equilibrated with the supernatant containing sheep growth hormone obtained in Example 3 with 1 M urea 5 mM ammonia carbonate buffer solution (pH 9.6) Both growth hormones were attached by passing at a rate of 2 / hour, and the materials and urea remaining free in the column were washed with 5 mM ammonia carbonate solution (pH 9.6). The protein attached to the resin was eluted with 300 ml of 5 mM ammonia carbonate solution (pH 9.6) and 300 ml of 0.5 M NaCl 5 mM ammonia carbonate solution in a straight line gradient, where both growth hormones were dissolved at a concentration of 0.1 M-0.2 M NaCl. Eluted.
[실시예 5]Example 5
S-200 겔 투과 크로마토그래피 과정S-200 gel permeation chromatography process
DE52 이온교환수지 크로마토그래피에서 얻은 양 성장 호르몬 용액 120ml을 YM10 한외 여과막 농축기(Amicon사, 미국)로 20ml가지 농축하거나, 황산암모니아(ammonium sulfate)를 30% 포화 용액까지 첨가하여 양 성장 호르몬을 침전시킨 후, 원심분리하여 침전물 0.25M NaCl 10mM 탄산암모니아 (pH 9.6) 20ml에 용해시키고 0.25M NaCl 10mM 탄산암모니아(pH 9.6)로 평형된 S-200 수지(Pharmacia, 스웨덴)컬럼(5cm×100cm)에서 10ml/cm2/hr 유속으로 통과시켜 단백질을 분리하였다.120 ml of sheep growth hormone solution obtained from DE52 ion exchange resin chromatography was concentrated in 20 ml of YM10 ultrafiltration membrane concentrator (Amicon, USA), or ammonium sulfate was added to 30% saturated solution to precipitate sheep growth hormone. After centrifugation, the precipitate was dissolved in 20 ml of 0.25 M NaCl 10 mM ammonia carbonate (pH 9.6) and 10 ml in an S-200 resin (Pharmacia, Sweden) column (5 cm × 100 cm) equilibrated with 0.25 M NaCl 10 mM ammonia carbonate (pH 9.6). Proteins were isolated by passing at a flow rate of / cm 2 / hr.
[실시예 6]Example 6
생물활성도 측정Bioactivity Measurement
양 성장 호르몬의 생물 활성도 측정은 뇌하수체 제거 수술을 받은 생후 4-5주일된 암컷 쥐(혈통 : Wister)를 Charles River Lab(미국)으로부터 구입하여 실시하였다. 상기 정제 방법에서 얻어진 양 성장 호르몬용액을 발열물질(pyrogen) 흡착 필터(CUNO사, 미국)를 통과시켜 발열물질을 제거하고 사용하였다. 뇌하수체를 제거시켜 성장 호르몬이 없는 쥐에게 양 성장 호르몬을 피하 주사하면서 체중을 측정, 몸무게가 증가함을 관찰하였다. 매일 다섯마리 쥐에게는 양 성장 호르몬이 0.2mg/ml 농도가 함유된 생리식염수를 0.5ml씩 주사하였고, 다섯마리의 쥐에게는 성장 호르몬이 함유되지 않은 생리 식염수 0.5ml씩을 14일간 주사하였다. 양 성장 호르몬을 주사한 쥐는 1일 평균 1.4%의 체중이 증가하여 2주일동안 23g(처음 체중의 20%)이 증가하였다(제5도), 이로써, 본 발명의 방법으로 정제된 양 성장 호르몬이 고도의 생물활성도를 가지고 있음을 알수 있다.Biological activity measurement of sheep growth hormone was carried out by purchasing from Charles River Lab (USA) 4-5 week old female rats (bloodline: Wister) undergoing pituitary removal. The positive growth hormone solution obtained in the purification method was passed through a pyrogen adsorption filter (CUNO, USA) to remove the exothermic material and used. The pituitary gland was removed and subcutaneously injected with sheep growth hormone to mice without growth hormone, and the weight was measured. Each day, five rats were injected with 0.5 ml of saline containing 0.2 mg / ml of sheep growth hormone, and five rats were injected with 0.5 ml of saline containing no growth hormone for 14 days. Mice injected with sheep growth hormone gained an average body weight of 1.4% per day, increasing 23 g (20% of the initial body weight) for 2 weeks (figure 5). It can be seen that it has a high biological activity.
Claims (5)
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019910024294A KR940005594B1 (en) | 1991-12-24 | 1991-12-24 | Method of refinning o-growth hormones |
| NZ245511A NZ245511A (en) | 1991-12-24 | 1992-12-18 | Ovine growth hormone (ogh) gene useful for mass production of ogh in e.coli, its production and purification |
| AU30238/92A AU653458B2 (en) | 1991-12-24 | 1992-12-18 | Ovine growth hormone gene and an expression thereof in E. coli |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019910024294A KR940005594B1 (en) | 1991-12-24 | 1991-12-24 | Method of refinning o-growth hormones |
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| Publication Number | Publication Date |
|---|---|
| KR930013105A KR930013105A (en) | 1993-07-21 |
| KR940005594B1 true KR940005594B1 (en) | 1994-06-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019910024294A KR940005594B1 (en) | 1991-12-24 | 1991-12-24 | Method of refinning o-growth hormones |
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| Country | Link |
|---|---|
| KR (1) | KR940005594B1 (en) |
| AU (1) | AU653458B2 (en) |
| NZ (1) | NZ245511A (en) |
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| KR100400638B1 (en) * | 2000-12-06 | 2003-10-08 | 주식회사 엘지생명과학 | Method for separating variants of recombinant human growth hormone |
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|---|---|---|---|---|
| JPS6011557A (en) * | 1983-04-19 | 1985-01-21 | ジエネツクス・コ−ポレイシヨン | Clone sheep growth hormone gene |
| CA1267615A (en) * | 1984-08-27 | 1990-04-10 | Dan Hadary | Method for recovering purified growth hormones from genetically engineered microorganisms |
| AU647374B2 (en) * | 1988-09-29 | 1994-03-24 | Natinco Nv | Sheep growth hormone |
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1991
- 1991-12-24 KR KR1019910024294A patent/KR940005594B1/en not_active IP Right Cessation
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| Publication number | Publication date |
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| AU3023892A (en) | 1993-07-01 |
| KR930013105A (en) | 1993-07-21 |
| AU653458B2 (en) | 1994-09-29 |
| NZ245511A (en) | 1994-04-27 |
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