Saccharomyces cerevisiae에서 인체 Lipocortin-1의 유전자 발현 및 분비

손정훈,나도선,이상기,Sohn, Jung-Hoon,Na, Doe-Sun,Rhee, Sang-Ki 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5

Phospholipase A2의 저해 단백질로서 항염증 치료제로 알려진 인체 lipocortin을 효모로부터 생산하기 위해 인체 lipocortin-1 유전자의 cloning 및 발현 연구를 수행하였다. 인체 lipocortin-1 cDNA를 CYC1, GALl-GALlO 및 PHO5 유전자의 promoter를 갖는 발현 vector에 cloning시킨 후 유전자의 발현을 조사한 결과 각 promoter의 강도 및 사용한 plasmid의 copy수에 따라 발현효율이 달라져 GALl-GALlO promoter와 $2{\mu}m$ 기원의 multicopy수의 plasmid를 사용했을 경우 인체 lipocortin-1 유전자의 발현효율이 가장 높음이 확인되었다. 효모 내에서 생산된 인체 lipocortin을 배지로 분비시키기 위해 효모의 ${\alpha}$ 교배인자의 pre-pro leader 배열과 인체 lipocortin-1 유전자를 융합시킨 결과 효율적인 post-translational processing을 거쳐 인체 lipocortin만이 일부 분해된 상태로서 체외로 분비됨이 관찰되었다. Human lipocortin is a phospholipase A2 inhibitory protein and known as a potential antiinflammatory agent. Thus, molecular cloning and expression of human lipocortin-1 gene was tried using a number of yeast expression vectors. cDNA of human lipocortin-1 was placed under the control of CYC1, GAL1-GAL10 and PHO5 promoters. The expression levels were varied according to the promoter strength and the plasmid copy number. It was observed that the human lipocortin-1 gene was able to be expressed under the control of all promoters employed but the highest level of gene expression was achieved by use of the divergent GAL1-GAL10 promoter. The $2{\mu}m$ based multicopy number plasmid showed an increased expression level by 50% compared with ARS based single copy number plasmid. To excrete human lipocortin produced in Saccharomyces cerevisiae cells into the culture medium, the pre-pro leader sequence of yeast a mating factor gene was fused with human lipocortin-1 gene. The efficient secretion of the native protein was observed with some extent of degradation in the culture medium after the post-translational processing of the fused protein.

재조합 효모를 이용한 Hirudin 발효생산조건의 최적화

이동훈(Dong-Hoon Lee),서진호(Jin-Ho Seo),손정훈(Jung-Hoon Sohn),최의성(Eui-Sung Choi),이상기(Sang-Ki Rhee) 한국생물공학회 1994 KSBB Journal Vol.9 No.1

Saccharomyces cerevisiae 에서 인체 Lipocortin - 1 의 유전자 발현 및 분비

손정훈,나도선,이상기 ( Jung Hoon Sohn,Doe Sun Na,Sang Ki Rhee ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.5

Human lipocortin is a phospholipase A2 inhibitory protein and known as a potential antiinflammatory agent. Thus, molecular cloning and expression of human lipocortin-1 gene was tried using a number of yeast expression vectors. cDNA of human lipocortin-1 was placed under the control of CYC1, GAL1-GAL10 and PHO5 promoters. The expression levels were varied according to the promoter strength and the plasmid copy number. It was observed that the human lipocortin-1 gene was able to be expressed under the control of all promoters employed but the highest level of gene expression was achieved by use of the divergent GAL1-GAL10 promoter. The 2 ㎛ based multicopy number plasmid showed an increased expression level by 50% compared with ARS based single copy number plasmid. To excrete human lipocortin produced in Saccharomyces cerevisiae cells into the culture medium, the pre-pro leader sequence of yeast a mating factor gene was fused with human lipocortin-1 gene. The efficient secretion of the native protein was observed with some extent of degradation in the culture medium after the post-translational processing of the fused protein.

한국산 거머리로부터 항혈전단백질의 검색과 분리 , 정제

이상권,손정훈,최의성,이상기 ( Sang Kwon Lee,Jung Hoon Sohn,Eui Sung Choi,Sang Ki Rhee ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Two Korean leech species, Hirudo nipponia and Whitmania sp., were examined for the presence of anticoagulant activity. Crude extracts of the leech head parts were fractionated by ethanol and the anticoagulant activity of these extracts was assessed with respect to the inhibition of thrombin, factor Xa and trypsin using the chromogenic substrate assay. While the extract from Hirudo nipponia inhibited all three serine proteases employed in the assay, that of Whitmania sp. inhibited only trypsin, but not thrombin and factor Xa. Thus, the extract from Hirudo nipponia which shows antithrombin activity was subjected to further purification. Three steps of column chromatography including gel filtration on Superose 12 column, anion exchange on Mono-Q and reverse phase HPLC on C-8 column could resolve 3 antithrombin activity peaks from anti-trypsin and anti-factor Xa activities, indicating the presence of multiple forms of thrombin-specific inhibitors in Hirudo nipponia. N-terminal amino acid sequences of the two of these forms showed a limited degree of homology with a portion of heparin cofactor Ⅱ.

학술발표회 초록 : ( 생물화공 (1) ) : 단백질 공학을 이용한 항혈전 단백 Hirudin 의 금속 친화성 분리정체

추창웅,장용근,손정훈,최의성,이상기,박영훈,정봉현 ( Chang Woong Chu,Yong Keun Chang,Jung Hoon Sohn,Eui Sung Choi,Sang Ki Rhee,Young Hoon Park,Bong Hyun Chung ) 한국화학공학회 1992 추계학술발표회 Vol.- No.-

한국산 거머리로부터 항혈전단백질의 검색과 분리.정제

이상권,손정훈,최의성,이상기,Lee, Sang-Kwon,Sohn, Jung-Hoon,Choi, Eui-Sung,Rhee, Sang-Ki 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

한국산 거머리인 Hirudo nipponia와 Whitmania sp.의 추출액으로 trypsin, factor Xa, thrombin에 대한 저해활성을 조사하였다. Hirudo nipponia로부터 얻은 추출액은 $20{\mu}g/ml$ 농도에서 이 효소들의 활성을 완전히 저해하는 반면 Whitmania sp.의 추출액은 동일조건하에서 trypsin만을 저해하였다. 이 Hirudo nipponia의 추출물로부터 항thrombin 인자를 분리 정제하기 위하여 anion exchange chromatography, size exclusion chromatography 및 HPLC를 이용하였고 최종적으로 세 종류의 단백질(I-A,I-B,I-C)을 얻었다. 이 중 두 종류 단백질(I-A, I-B)의 아미노말단 서열을 부분적으로 분석한 결과 이 단백질이 주로 산성 아미노산잔기로 구성되어 있으며 heparin cofactor II의 일부분과 유사성이 있음이 발견되었다. Two Korean leech species, Hirudo nipponia and Whitmania sp., were examined for the presence of anticoagulant activity. Crude extracts of the leech head parts were fractionated by ethanol and the anticoagulant activity of these extracts was assessed with respect to the inhibition of thrombin, factor Xa and trypsin using the chromogenic substrate assay. While the extract from Hirudo nipponia inhibited all three serine proteases employed in the assay, that of Whitmania sp. inhibited only trypsin, but not thrombin and factor Xa. Thus, the extract from Hirudo nipponia which shows antithrombin activity was subjected to further purification. Three steps of column chromatography including gel filtration on Superose 12 column, anion exchange on Mono-Q and reverse phase HPLC on C-8 column could resolve 3 antithrombin activity peaks from anti-trypsin and anti-factor Xa activities, indicating the presence of multiple forms of thrombin-specific inhibitors in Hirudo nipponia. N-terminal amino acid sequences of the two of these forms showed a limited degree of homology with a portion of heparin cofactor II.

유전자 재조합에 의해 제조된 하루딘의 항응고 작용

정기화,김영식,이상기,손정훈,최의성,엄은미,정정숙,정춘식 한국응용약물학회 1993 Biomolecules & Therapeutics(구 응용약물학회지) Vol.1 No.2

Hirudin is a potent inhibitor of thrombin, which was originally obtained from the medicinal leech (Hirudo medicinalis). Now it is being produced through the recombinant technology on a large scale. Recombinant hirudin has been assayed for the anticoagulant activity by the measurement of clotting time and the inhibition of thrombin actvity using a chromogenic substrate. The assay range of partial thromboplastin time and thrombin time is within 0.2∼1.0 ,㎍/㎖. Thrombin time is more sensitive to the measurement of clot. Ex vivo study showed the level of hirudin in rat plasma was highest in 10 min and then it was eliminated slowly. The half-life of r-hirudin was 80∼110 min depending on the assay methods. Intraveneous injection of russel viper venom was used for thrombus induction combined with vena cava ligation. Inhibition of venous thrombosis was observed with i.v. hirudin. It was dependent on the concentration of hirudin.

메타놀 자화 효모 Hansenula polymorpha를 이용한 재조합 인체 표피 성장인자 유전자의 발현 및 분비

오용익,송정훈,최의성,김희철,이상기 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.5

인체 표피세포의 성장을 촉진하고 위산의 분비를 억제하는 인자인 인체 EGF(human Epidermal Growth Factor)의 유전자를 메타놀 자화 효모인 Hansenula polymorpha를 이용하여 발현시키고 이를 배지내로 효율적으로 분비시키기 위해서 H. polymorpha의 methanol oxidase(MOX) 유전자의 codon usage에 따라 이 유전자를 화학적으로 합성하였다. 메타놀에 의해 발현 유도되는 강력한 promoter인 MOX promoter와 S. cerevisiae의 교배인자 α의 분비 신호인 pre-pro leader 서열을 이용하여 인체 EGF의 발현 및 분비 vector를 제조하였으며 이 vector가 효과적으로 숙주세포의 염색체 DNA에 도입됨을 확인하였다. 재조합 H. polymorpha는 소변 유래의 인체 EGF와 동일한 epitope을 지니는 재조합 인체 EGF를 효과적으로 발현시켰고 이를 세포외로 분비하여 준최적화 배양조건에서 최대 24 ㎎/l 의 생산수율을 나타내었다. Using a methylotrophic yeast Hansenula polymorpha, a heterologous gene expression and secretion system was developed for the production of hEGF(human Epidermal Growth Factor) which has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The hEGF gene was chemically synthesized according to the preferred codon usage in H. polymorpha and expressed under the control of the strong and inducible methanol oxidase(MOX) promoter. The mating factor α pre-pro leader sequence of Saccharomyces cerevisiae was employed for hEGF to be secreted into the extracellular medium. This expression cassette was stably integrated into the host chromosomal DNA. Mature hEGF was efficiently expressed and secreted into the extracellular medium. About 24 ㎎/l of hEGF was detected in the cuture supernatant of a transformant with pA-EGF3 under the suboptimal culture conditions.