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- 저자 : 함경수(Kyung So (검색결과 4 건)
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E . coli 에 의해 생산된 재조합 사람 프리트롬빈 2 의 정제 및 활성화
소인섭,이승철,김수원,함경수,김지영 ( In Seop So,Seung Cheol Lee,Soo Won Kim,Kyung Soo Hahm,Ji Young Kim ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.1
Human prethrombin 2 was purified from a recombinant E. coli cells in which human prethrombin 2 was produced as insoluble aggregates, amounting to 5 to 10% of the total bacterial proteins. Isolation of inclusion bodies and the subsequent solubilization of the insoluble proteins in a denaturing solution containing sodium dodecyl sulfate and dithiothreitol yielded good recovery of prethrombin 2 with at least 75% purity. Prethrombin 2 was further purified by two passages of Sephacryl S-200 gel chromatography. The purified prethrombin 2 was reacted with the polyclonal antibodies against human prothrombin. The N-terminal sequence of the prethrombin 2 was same as that of the native human prethrombin 2. The purified prethrombin 2 was renatured by stepwise removal of the denaturants by slow dialysis. The renatured prethrombin 2 was activated to thrombin by ecarin, a prothrombin activator from Echis carinatus and the thrombin activity generated was detected by a chromogenic assay. This study showed that recombinant prethrombin 2 produced in E. coli could be converted to thrombin.
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함준수(Joon Soo Hahm),박경근(Kyung Geun Park),박준용(Joon Yong Park),한동수(Dong Soo Han),이민호(Min Ho Lee),기춘석(Choon Suhk Kee),박경남(Kyung Nam Park),이광수(Kwang Soo Lee),최은아(Eun A Choi),이명규(Myung Kyo Lee),함경수(Kyung So 대한소화기학회 1996 대한소화기학회지 Vol.28 No.1
N/A Background/Aims: Biliary proteins have been suggested to play an important role in nucleation and gallstone formation. However, the exact roles and characteristics have not been completely documented. The aim of the present study is to isolate and characterize the nucleating protein extracted from gallstones. Methods: We tried to extract, isolate and characterize proteins in patients with gallstones. Twenty-two gallstones were obtained(12 cholesterol, 10 pigment) at cholecystec- tomy and extracted with ethanol/ether mixture. Then, isoelectric focusing was performed and gallstone proteins were analysed by SDS-polyacrylamide gel electrophoresis. The amino acids were also analysis by the autoamimo acid analyzer system. Results: The mean amount of gallstone protein was 4.15 mg/(g stone) in cholesterol and 16.15 mg/(g stone) in pigment stone. The proteins from both cholesterol and pigment stones showed major bands at low pH on isoelectric focusing. On SDS-PAGE, low molecular protein bands were noted, mainly below 45KD in both cholesterol and pigment stones. The composition of aspartate and glutamate was 21.6% in cholesterol stones and 22.7% in pigment stones. Conclusions: The proteins in cholesterol and pigment gallstones are low molecular weight acidic proteins, and these acidic proteins seem to play an important role in the pathogenesis of gall stones. However, it remains to be determined whether these proteins differ in functional roles from different gallstones. (Korean J Gastroenterol 1996;28: 92 - 100)
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Rana Box을 포함한 Brevinin-1 및 Thanatin의 구조-항생활성 상관관계
신송엽,강주현,이동건,장소윤,서무열,함경수 한국산업미생물학회 1999 한국미생물·생명공학회지 Vol.27 No.6
Brevinin-1 및 thanatin의 카르복실말단의 Rana box의 아미노산 서열 및 thanatin의 꼬리부분의 아미노산 서열이 항생활성에 미치는 영향을 조사하기 위하여, thanatin 및 brevinin-1 의 Rana box위치에 아미노산 서열변화를 유도한 펩타이드를 고상법에 의하여 합성하고 이들의 항균, 항진균, 항암활성 및 음이온성 인지질막 응축활성을 측정하였다. Brevinin-1보다 thanatin의 Rana box의 염기성서열이 항균 및 항진균활성에 더 중요한 영향을 주었으며, thanatin의 꼬리 서열인 QRM은 항균 및 항진균 활성에 모두 중요한 역할을 담당한다는 사실을 알았다. Brevinin-1의 항암활성 및 인지질 응축활성에는 Rana box서열의 서열보다는 소수성 잔기가 많이 포함된 긴 아미노말단의 서열이 중요하다는 사실을 알았다. 세포막을 모방하는 조건인 30 mM SDS용액에서 thanatin의 Rana box에 brevinin-1의 서열을 도입(T-B1)하고 T-B1유사체에서 thanatin의 꼬리서열을 제거함(T-B2)으로서 α-helicity가 증가함을 보였다. Brevinin-1은 같은 조건에서 전형적인 α-helical structure를 나타내었으며 brevinin-1의 Rana box에 thanatin의 서열을 도입(B-T유사체)함으로서 α-helicity가 감소함을 보였다. 즉 B-T1 및 B-T2에서의 α-helicity의 증가는 항균, 항진균 및 항암활성에 영향을 미치지 않았으나, B-T에서의 α-helicity의 감소는 항균활성의 감소에 영향을 미치는 것으로 보아 brevinin-1의 α-helicity는 항생활성에 중요한 역할을 한다는 사실을 알았다. 또한 thanatin이 brevinin-1에 비하여 높은 β-lactamase 방출효과를 나타내는 것으로 보아 thanatin의 E. coli에 대한 항균활성은 세포벽에 대한 파괴효과에 의한 것으로 여겨진다. In order to investigate structure-antibiotic activity relationships of brevinin-1 and thanatin containing Rana box composed of basic loop formed by disulfide bridge in their carboxy terminus, thanatin, brevinin 1 and their analogues (T-B1, T-B2 and B-T) in which their Rana box sequence exchanged was designed and synthesized by the solid phase method using Fmoc-chemistry. The basic sequence of Rana box of thanatin had more significant effect on both antibacterial and antifungal activity than that of brevinin 1. The tail sequence (QRM) of thanatin was found to be important in its antibacterial and antifungal activity. Rana box sequence of brevinin-1 did not have a significant effect on its antitumor and phospholipid vesicle-aggregating activities. Brevinin-1 showed stronger α-helical structure in the membrane-mimicking environment such as SDS micelle than thanatin. A remarkable increase in a-helicity of T-B1 and T-B2 caused a significant reduction on antibacterial and antifungal activities. In contrast, a little decrease in α-helicity of B-T caused 4-fold reduction on antibacterial activity. These results suggested that the α-helicity of bervinin-1 plays more important role in antibiotic activity than that of thanatin. Furthermore, antibacterial activity of thanatin against E. coli resulted from the disruptive effect against the outer cell membrane of E. coli.
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