대장균에서 재조합 Rat Guanine deaminase 유전자의 발현, 정제 및 분석

성연선,곽상준,박대성,김향원,이희영 단국대학교 1998 論文集 Vol.33 No.-

Guanine deaminase(EC, 3.5.4.3, ;Guanine aminohydrolase, GAH, GDA) catalyzes the deamination reaction of guanine to xanthine irreversibly. The cDNA encoding rat guanine deaminase had been isolated from a λZAPII rat brain expression library using antibody generated against purified rat guanine deaminase. Toe obtain recombinant GDA and analyze the property of catalytically conserved retion, here we expressed the recombinant GDA in E.coli and showed the retention of its catalytic activity similar to native rat GDA. To make the construct carrying coding region to GDA in pGEX4T prokaryotic expression vector, the region encompassing open reading frame of pBlue-GDA was PCR amplified and subcloned into pGEX4T prokaryotic expression vector with correct reading frame of fusion carrier glutathione S-transferase. After transformation to E.coli DH5a. The bacteria carrying pGEX-GDA was grwon in the condition of IPTG induction. The fusion protein GST-GDA was purified with GSH-sepharose affinity chromatography. The purified GST-GDA was subsequently digested with biotin-conjugated thrombin, and thrombin was removed by streptavidin agarose. Then sample were treated with GSH-sepharose affinity chromatography to remove the contaminant GST and GST-GDA. The purified recombinant GDA showed 70% of specific activity relative to that of purified rat GDA. The recombinant GDA showed identical molecular weight with rat GDA in SDS-PAGE.

트란스페린에 대한 단클론항체의 생산과 방사면역측정법의 개발

한현식,곽상준 충북대학교 의과대학 충북대학교 의학연구소 1997 忠北醫大學術誌 Vol.7 No.1

트란스페린은 주로 간에서 생산되는 금속결합 운반 당단백으로 주요한 역할은 헤모글로빈의 합성을 위하여 철을 운반하는 것이다. 근래 트란스페린의 철공급 외의 기능으로 감염에 대한 숙주 저항에 관한 견해들이 있다. 현재 트란스페린을 직접 측정하는 방법인 방사면역 확산법은 혈장의 농도를 측정하는 데에는 문제가 없으나 체액에 미량 존재하는 부위의 측정에는 민감도가 떨어진다. 본연구에서는 트란스페린에 대한 단클론항체를 만들어 이들의 특성을 분석하고, 이를 이용한 민감한 측정법을 개발하며, 나아가 트란스페린치와 숙주저항성과의 관련성을 보고자 하였다. 단글론항체를 3가지 얻었는데, 이들은 모두 IgG₁이었고 이항체는 트란스페린의 철결합 유무에 관계 없이 같은 정도로 결합하였다. 트란스페린의 항원결정기는 서로 같거나 아주 근접하여 위치하는 것으로 생각 된다. 트란스페린을 방사성옥소로 표식하고, 폴리스타이린관을 고상으로 이용한 면역측정법을 개발하였는데, 검색구간은 0.1 - 20 ㎍/㎖ 이었고, 정밀도 및 분석적 회수율이 높았다. 혈청은 1,000배로 희석하여 측정하고, 소변은 희석하지 않고 측정할 수 있었다. 급성 감염성 질환과 비염증성 질환에서 얻은 혈청과 소변의 트란스페린치를 측정한 결과 유의한 차이를 보이지는 않았다 Transferrin is a metal-binding transport glycoprotein mainly produced in the liver. In addition to the iron supplying function, host resistance to infection of transferrin has been recently proposed. Transferrin is currently measured by radial immunodiffusion, but its sensitivity is low, especially for assaying body fluid other than serum. For developing a more sensitive assay system, monoclonal antibodies to human transferrin were produced, all of which are of the IgG₁subclass. These monoclonal antibodies bound transferrin to the same degree regardless of iron binding. The epitopes of transferrin seemed to be nearly the same or very close in position, so that competitive binding immunoassay system was developed in which transferrin was labelled with I125 and polystyrene tube was used as a solid phase. The detectable rang of transferrin by the assay system was between 0.1 and 20㎍/ml. The reproducibility and the recovery were satisfactory Serum was assayed after diluting 1 to 1000, but urine was assayed without dilution. The transferrin levels of acute infectious subjects were not significantly different from those of non-inflammatory subjects in urine as well as serum.

Modulation of detoxifying Potential and Differential Expression of Glutathione Transferase Isoenzyme in the Bacterial ada Gene-transfected Murine Hepatoma Cell

Kwak, Sahng June,Lim, In Kyoung,Kimm, Seung Won,Park, Sang Chul Korean Society of Molecular Biology 1993 Molecules and cells Vol.3 No.3

Session F : Eukaryotic Gene Structure and Expression : Modulation of detoxifying potential by differential expression of glutathione transferase isoenzyme genes in bacterial ada transfected rat hepatoma cells

Sahng June Kwak,In Kyoung Lim,Gyung Ho Choi,Jee Young Choi,Seung Won Kim,Sang Chul Park 한국생화학분자생물학회 (구 한국생화학회) 1993 추계학술대회집 Vol.- No.-

Histone Lysine Methylation

곽상준,Kwak, Sahng-June Korean Society of Life Science 2007 생명과학회지 Vol.17 No.3

유핵세포의 게놈(genome)은 단백-DNA복합체인 염색질(chromatin)의 형태로 존재하는데, 생명현상을 유지하기 위해서는 생명체 또는 세포가 처한 상황에 맞게 염색질의 구조를 변화시키는 역동적인 조절기전이 필요하다. 염색질을 구성하는 기본단위는 히스톤 8량체 (histone octamer)를 포함하는 뉴클레오좀(nucleosome)이다. 히스톤 단백에는 여러 종류의 공유결합성 수식이 일어나는데, 그 중 하나가 라이신 잔기(lysine residue)에 일어나는 메틸화이다. 최근 수년간의 연구로 여러 개의 히스톤 라이신 메틸화효소(histone lysine methyltransferase, HKMT), 이에 결합하는 염색질단백 및 메틸화와 관련된 후생유전학적 현상이 밝혀졌으며, 특히 정밀한 연구방법을 동원한 다방면의 실험을 통하여 비록 자세한 기전과 전체적인 윤곽의 규명은 미흡하더라도 라이신 메틸화가 후생유전학적 변화를 초래하는 일부 과정이 규명 되었다. 또한 여러 종류의 라이신 탈메틸화효소가 최근에 발견됨에 따라, 아세틸화, 인산화등 다른 공유결합성 수식보다는 상대 적으로 안정되더라도, 히스톤 메 틸화로 유발되는 후생유전학적 변화가 불가역성이 아님을 알게 되었다. Our genome exists in the form of chromatin, and its structural organization should be precisely regulated with an appropriate dynamic nature for life. The basic unit of chromatin is a nucleosome, which consists of a histone octamer. These nucleosomal histones are subject to various covalent modifications, one of which is methylation on certain lysine residues. Recent studies in histone biology identified many histone Iysine methyltransferases (HKMTs) responsible for respective lysine residues and uncovered various kinds of involved chromatin associating proteins and many related epigenetic phenotypes. With the aid of highly precise experimental tools, multi-disciplinary approaches have widened our understanding of how lysine methylation functions in diverse epigenetic processes though detailed mechanisms remain elusive. Still being considered as a relatively more stable mark than other modifications, the recent discovery of lysine demethylases will confer more flexibility on epigenetic memory transmitted through histone lysine methylation. In this review, advances that have been recently observed in epigenetic phenotypes related with histone lysine methylation and the enzymes for depositing and removing the methyl mark are provided.

Aging process is accompanied by increase of transglutaminase C

Park, Jeong Soo,Kwak, Sahng June,Choi, Jee Young,Yeo, Eui Ju,Kim, Kyung Ok,Seong, Sang Cheol,Hwang, Yong Chul,Park, Sang Chul,Park, Yong Hoon,Han, Jeong A 한림대학교 한림과학원 부설 환경ㆍ생명과학연구소 1998 국제학술회의 Vol.1998 No.-

Crosslinking has been suggested as one of the mechanisms involved in the aging process. Among the various random or enzyme-mediated crosslinking reactions, transglutaminase (TGase)-catalyzed crosslinking activity has been proposed for its possible involvement in cell proliferation, differentiation, carcinogenesis, programmed death and aging. Moreover, recent findings of TGase C as a putative signal transducer and cell cycle regulator has renewed interest in the study of TGase C in relation to aging phenomena. The ubiquitous presence of TGase C compared to the organ-specific localization of other types of TGases has attracted special attention as a cellular aging device. In the present study, for in vitro studies we have analyzed the comparative pattern of TGase C in young and old human red blood cells, separated by density defferentiation, and in early and late-passage of hydrogen peroxide-treated human primary fibroblasts. For in vivo study, we monitored the age-dependent changes of TGase C in the liver and brain tissues of 4, 12, 18, and 24-month-old Sprague-Dawley rats. We obtained evidence that both the activity and protein levels of TGase C were high in old RBC and late-passage of hydrogen peroxide-treated vibroblasts. Similar findings were seen in liver and brain tissue such as age-dependent increases in TGase activity and protein level in an organ-specific pattern. These data suggest that TGase C might play an active role in the cellular process with age.

Mechanism of Drug Resistance in Hepatoma tissues : Differential Expression of Gamma-gluamyl Transpeptidase, Glutathione Transferase and P-glycoportein Genes in vitro and in Vivo

Kim, Eung Gook,Kwak, Sahng June,Park, Sang Schul,Kim, Soo Tae Korean Society of Molecular Biology 1991 Molecules and cells Vol.1 No.2

Transcriptional Control of the glnD Gene Is Not Dependent on Nitrogen Availability in Escherichia coli

Kim, In-Hoo,Kwak, Sahng June,Kang, Jungog,Park, Sang Chul Korean Society of Molecular Biology 1998 Molecules and cells Vol.8 No.4

Multiplex PCR-aided Differential Diagnosis of Taeniid Species

Hye-Jung Lee(이혜정),Min Seo(서민),Sahng-June Kwak(곽상준) 한국생명과학회 2010 생명과학회지 Vol.20 No.6

아시아조충과 무구조충의 편절은 형태학적으로 유사해 감별진단하기가 쉽지 않다. 하지만 아시아조충의 경우 감염자에서 낭미충증 등의 심각한 합병증을 일으킬 가능성을 배제할 수 없으므로 두 기생충의 정확한 진단이 필요하다. 최근 기생충학 분야에서 DNA 서열에 기초한 진단 방법이 널리 쓰이고 있다. 본 연구에서는 다중 중합 효소연쇄반응을 이용해 한국인에서 발견된 태니아 속 조충류의 감별진단을 시도해 보고자 했다. 중합효소연쇄반응을 위해 Ta4978F, Ts5058F, Tso7421F, Rev7915 4개의 시발체(프라이머)를 사용했으며, 그 결과 태니아의 종 동정이 정확하고 신속하게 이루어질 수 있었다. 중합효소연쇄반응 방법을 도입한다면 한국에서 인체 태니아 조충의 역학적 소견을 용이하게 재검토할 수 있으리라고 생각한다. Differential diagnosis of the taeniid proglottids between Taenia asiatica and T. sagniata is a daunting task due to their close morphological similarity. However, to correctly diagnose them on time is important in managing infected patients, as well as for reducing serious complications such as cysticercosis. Currently, DNA-based methods for the dissection of genomic information of parasites are being employed to make accurate and rapid diagnoses in the field of parasitology. In this study, multiplex PCR was established and exploited to identify exact species of taeniid adult worms recovered from Korean people. To discriminate one from the other other, primers-Ta4978F, Ts5058F, Tso7421F, and Rev7915- were used for the multiplex PCR, which provided swift and precise identification of the taeniid worms being observed. Also,having instituted PCR methodology, we ascertained that easiness would be achieved to reassess and re-evaluate Korean endemic data on human taeniid cestodes.

Expression of Transglutaminase in Human Cervical Cancer Tissues and HPV - 16 Transfected Human Epithelial Cells

Park, Sang Chul,Kim, Soo Youl,Kwak, Sahng June,Kim, Eung Gook,Song, Kye Yong 한국유전학회 1988 Genes & Genomics Vol.10 No.4

Multiple forms of tranglutaminases(TGase) are present in human epidermis and the cultured human keratinocytes. In an effort to understand the regulation of epidermal TGase in vivo, enzymes in human foreskin tissues and in the cultured human keratinocytes, derived from the same tissue and modified with oncogenic insertion, were examined. The cultured human kerationcytes were inserted or transfected with SV40 DNA virus, kirsten RNA virus or human papilloma viral genes, resulting in immortalization or in tumorigenic transformation. TGases from the tissue or cells were fractionated by elution with 0.03 M NaCl in DEAE-cellulose column for TGase E activity and by elution with 0.5 M NaCl in the same DEAE-column for TGase C activity, while TGase B activity was monitored in the particulated fraction. TGase E activity was the major extractable enzyme activity of the in vivo skin tissue. However, this TGase E activity was negligibly detectable in the immortalized cell lines regardless of the tumorigenicity under the varying medium conditions. And among the immortalized cell lines (RHEK-1, KHOS/RHEK-1, PSV2-ras/RHEK-1, Ki/HPA-IA), activities of TGase C and B were most highest in RHEK-1 cell line, while those activities were decreased with increase of tumorigenicity. From these results, it could be concluded that TGase E activity is deeply related with terminal differentiation of the skin tissues and TGase B and C activities are modulated by the cellular status of transformation.