• Journal of Microbiology
• /
• 제45권6호
• /
• pp.593-596
• /
• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• 대한한의학회지
• /
• 제16권1호통권29호
• /
• pp.281-294
• /
• 1995

The effect of Sam Hwa San on the Na-K-ATPase activity was evaluated in microsomal fraction prepared from rabbit cerebral cortex to determine whether Sam Hwa San affects Na-K-ATPase activity of nervous system. Sam Hwa San markedly inhibited the Na-K-ATPase activity in a dose-dependent manner with an estimated $I_{50}$ of 0.12%. Optimal pH for the Na-K-ATPase activity was at 7.5 in the presence or absence of Sam Hwa San. The degree of inhibition by the drug more increased at acidic and alkalic pHs than neutral pH. Kinetic studies of substrate and cationic activation of the enzyme indicate classic noncompetitive inhibition fashion for ATP, Na and K, showing significant reduction in Vmax without a change in Km. Dithiothreitol, a sulfhydryl reducing reagent, partially protects the inhibition of Na-K-ATPase activity by Sam Hwa San. Combination of Sam Hwa San and ouabain showed higher inhibition than cumulative inhibition. These results suggest that Sam Hwa San inhibits Na-K-ATPase activity in central nervous system by reacting with, at least a part, sulfhydryl group and ouabain binding site of the enzyme protein, but with different binding site from those of ATP, Na and K.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.301-304
• /
• 2005

S-Adenosyl-L-Methionine(SAM) has an important role for DNA methylation and cell signaling. SAM was synthesized from methionine and ATP by SAM synthetase and play an pivotal function in the primary and secondary metabolism of cells. Recent studies have revealed in the effect of SAM in case of morphological differentiation in both eukaryotes and prokaryotes. We isolated SAM gene from Saccharomyces cerevisiae and cloned it into expression vector for E. coli respectively. An 1.15 kb SAM-s gene fragment was isolated by Low-strigency PCR using ORF primer. By the analysed primary sequence deduced from DNA sequence, this gene included conserved domains similar with other well-known SAM synthetase. First of all, SAM synthetase gene cloned pGEM-T vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. Typical characteristic analysis of this enzyme is underway.

• 대한한방부인과학회지
• /
• 제19권1호
• /
• pp.166-177
• /
• 2006

Purpose : This investigation was undertaken to evaluate the antiproliferation, atoptosis of Sam-nueng(Sparganii Rhizoma) extract using MCF-7 human breast cancer cells. Methods : MCF-7 cells were cultured in Dulbecco's modified Eagle's medium/F12(DMEM/F12) supplemented with 10 % fetal bovine serum(FBS; Gibco) and antibiotics. At varying times after extract treatment, cells were harvested with scraper and processed for analysis of protein expression, proliferation, cytotoxicity and apoptosis. Results : Our results show that the extract of Sam-nueng(Sparganii Rhizoma) strongly inhibits the proliferation of MCF-7 cells in a dose and time-dependent manner. Sulforhodamine B showed that the addition of Sam-nueng(Sparganii Rhizoma) extract reduced the viability of MCF-7 cells in a dose-dependent manner. Poly[ADP(ribose)] polymerase(PARP) which serves as a marker of cells undergoing apoptosis, a major substrate for caspase-3 was extensively cleaved in the Sam-nueng(Sparganii Rhizoma)-treated cells. Conclusion : So, we can conclude that Sam-nueng(Sparganii Rhizoma) can have an inhibitive effect on MCF-7 human breast cancer cells by variable mechanisms.

• 동국한의학연구소논문집
• /
• 제8권2호
• /
• pp.83-96
• /
• 2000

이 실험의 목적은 탄수화물 부하식이요법시의 삼출건비방 투여가 달리기 선수의 뇨중 대사성분변화에 미치는 영향에 대해 관찰 연구한 것이다. 탄수화물 부하식이시 삼출건비탕(蔘朮健脾湯) 투여는 전해질대사 관련 변인에 있어서 $Na^+$, $Cl^-$, $K^+$, $Ca^{+ +}$에 유의성 있는 영향을 미쳤으며, 이는 세포막 투과성의 변화를 의미하는 것으로 $Na^+$-$K^+$ pump의 활성도가 증가하여 운동수행능력을 향상시킬 것으로 기대되나 추후 전해질 대사와 관련하여 renin, aldosterone 등의 호르몬 분비 기전에 관해서도 지속적인 연구가 진행되어야 할 것으로 사려된다.

• BMB Reports
• /
• 제32권1호
• /
• pp.82-85
• /
• 1999

The movement protein of cucumber mosaic virus (CMV) is required for cell-to-cell movement of viral RNA. The movement of viral RNA occurs through the plant intercellular connection, the plasmodesmata. The viral movement protein was known to be multi-functional. In this work, a series of deletion mutants of CMV movement protein gene were created to identify the functional domains. The mutated movement proteins were produced as inclusion body in E. coli, and purified and renatured. A polyclonal antibody was raised against the CMV-Kor strain (Korean isolate) movement protein expressed in E. coli. The ability of the truncated proteins to bind to ssRNA was assayed by UV cross-linking and gel retardation analyses. The results indicate that the domain between amino acids 118 and 160 of CMV movement protein is essential for ssRNA binding.

• 한국당과학회:학술대회논문집
• /
• 한국당과학회 2006년도 제1회 연례학술대회
• /
• pp.44-44
• /
• 2006

• 한국당과학회:학술대회논문집
• /
• 한국당과학회 2006년도 제1회 연례학술대회
• /
• pp.42-43
• /
• 2006

• 한국동물학회:학술대회논문집
• /
• 한국동물학회 1997년도 한국생물과학협회 학술발표대회
• /
• pp.239.1-239
• /
• 1997

No Abstract, See Full Text

• 한국음향학회지
• /
• 제23권1호
• /
• pp.1-7
• /
• 2004

본 연구에서는 항원-항체 결합반응을 통해 액상의 단백질 분자를 감지할 수 있는 SH형 SAW센서를 개발하였다. 측정하고자 하는 항원으로는 anti-Human-immune-globulin (anti-HigG) 단백질을, 이에 대응하는 항체로는 HigG를 채택하였다. 압전 단결정 LiTaO₃를 사용하여 100 MHz로 발진되는 센서를 제작하고, 센서의 지연선 위에 Ti/Au층을 증착하였다. 증착된 Au층 위에 SAM (self assembled monolayer)을 형성시켜서 추가되는 항원의 농도에 의한 센서의 주파수 변화를 측정하였다. 개발된 센서의 최소검출단위는 노이즈 레벨 400 Hz 이하 값에 10.8 ng/ml/Hz의 민감도를 가지며, anti-HigG 항원의 질량하중 효과에 대해 안정적인 반응을 보였다.