새로운 유전자 재조합 방법을 이용한 대장균에서의 인간 tissue inhibitor of mtrix metalloproteinase-2 (TIMP-2) 유전자의 가용성 발현

김종욱,최동순,주현,민철기,Kim, Jong-Uk,Choi, Dong-Soon,Joo, Hyun,Min, Churl-K. 한국생물공학회 2008 KSBB Journal Vol.23 No.3

암세포의 침윤은 숙주 조직의의 기저막과 세포외 기질을 침투함으로써 일어난다. 침윤과 전이과정에는 단백질가수분해 효소인 matrix metalloproteinases (MMPs)가 깊이 연관되어 있는 것으로 알려져 있으며, MMP의 가수분해 활성은 tissue inhibitors of metalloproteinases (TIMPs)라는 억제 단백질에 의해 억제된다. TIMP-2는 21kDa 크기의 포유류 단백질로 대장균에서 과발현 시 다른 많은 포유류 단백질과 마찬가지로 가용성이 낮은 봉입체 형태로 발현된다. TIMP-2 단백질의 접힘에 6개의 이황화결합이 필요하고, 이는 일반적으로 대장균 환경은 적합하지 않다. 본 연구에서는 대장균에서 불가용성으로 발현되는 TIMP-2 유전자를 유전자셔플링 기법의 한 가지인 StEP (staggered extension process)를 변형하고 동시에 $Mn^{2+}$ 농도 변화와 dGTP 불균형을 이용한 무작위 돌연변이 기법을 혼용하여 대장균에서 가용성 TMP-2 재조합 변이체를 생성하고자 하였다. 무작위로 재조합된 TIMP-2 유전자 중에서 가용성으로 발현되는 TIMP-2 유전자를 선별하기 위해서 chloramphenicol acetyltransferase (CAT)-융합 방법을 도입하였다. CAT 유전자가 가용성으로 발현되는 재조합 TIMP-2 유전자에 융합되면 이를 갖는 E. coli는 높은 chloramphenicol 환경에서 생존이 가능하게 된다. 이러한 in virro mutageuesis 기법과 CAT-융합 방법으로 대장균 가용성 TIMP-2 재조합 변이체를 14가지 얻을 수 있었다. 변이체 TIMP-2의 아미노산서열 분석과 구조 분석 결과 주로 소수성 아미노산이 친수성 아미노산으로 전환되었고, MMP와의 결합이 관여하지 않는 C-말단 부위에 돌연변이가 집중되어 있었다. 본 연구에서 개발된 간편하고 새로운 in vitro 재조합 방법과 CAT을 이용한 스크리닝 기법은 다른 많은 대장균 내 불가용성 단백질의 발현에도 사용될 수 있을 것으로 사료된다. The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.

GnRH (Gonadotropin-Releasing Hormone)에 의한 자궁내막암 유래 세포주의 세포 증식 억제 기전에 있어서 Integrin, FAK (Focal Adhesion Kinase) 및 ERK (Extracellular Signal Regulated Kinase)의 역할

최종락,박동욱,최동순,민철기,Choi, Jong Rak,Park, Dong Wook,Choi, Dong Soon,Min, Churl K. 대한생식의학회 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.2

Objective: To investigate new signal transduction cascade through integrin, FAK and ERK in the suppressed cell proliferation by GnRH-I and -II. Method: Human endometrial cancer cells (HEC1A) were cultured under the following condition: DMEM/F12 (10% FBS). GnRH-I and -II were treated time (0, 5, 10, 15, 20, 30 min; 100 nM) and dose (10 nM or 100 nM; 20 min) dependent manner according to experimental purposes. Cell proliferation was measured using [$^3H$] thymidine incorporation assay. Immunoblotting was utilized to detect proteins. Results: GnRH-I and -II inhibited proliferation of HEC1A cells and induced expression of integrin ${\beta}3$. Phosphorylation of FAK and ERK were induced by GnRH-I and -II. Conclusion: GnRH inhibited cell proliferation via the expression of integrin and FAK, ERK phosphorylation. 목 적: 본 연구를 통해 GnRH 의한 세포 분열의 억제는 integrin, FAK 빛 ERK를 통한 세포 내 신호전달 기전을 통하여 일어남을 규명하고자 하였다. 연구방법: 연구에 사용된 인간자궁내막암 세포주는 DMEM/F12 (10% FBS)의 조건에서 배양 하였다. GnRH-I과 -II는 실험 목적에 따라 100 nM 농도로 0, 5, 10, 15, 20, 30분간 또는 10 nM or 100 nM의 농도로 20분간 처리 하였다. 세포의 분열 정도는 [$^3H$] thymidine incorporation assay를 이용하여 정량적으로 측정 하였으며, Immunoblotting 방법을 이용하여 단백질의 발현을 확인 하였다. 결 과: GnRH-I과 -II 모두 HEC1A 세포의 세보분열을 억제하였으며 integrin ${\beta}3$의 발현을 증가 시켰다. GnRH-I과 -II를 처리 후 FAK 및 ERK의 안산화가 증가됨을 관찰할 수 있었다. 결 론: GnRH에 의한 세포분열의 억제는 integrin의 발현과 FAK 및 ERK의 인산화 과정을 통하여 일어남을 알 수 있었다.

TGF-β1에 의하여 유도된 인간자궁내막의 탈락막화(Decidualization)에 있어서 ERK (Extracellular Signal Regulated Kinas)와 PPARγ (Peroxisome Proliferator-Activated Receptor Gamma)의 역할

장혜진,이재훈,김미란,황경주,박동욱,민철기,Chang, Hye Jin,Lee, Jae Hoon,Kim, Mi Ran,Hwang, Kyung Joo,Park, Dong Wook,Min, Churl K. 대한생식의학회 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.2

목 적: 본 연구를 통해 $TGF-{\beta}1$에 의해 유도된 인간자궁내막의 탈락막화 과정에서 ERK와 $PPAR{\gamma}$의 역할을 규명하고자 하였다. 연구방법: 자궁내막 기질세포는 DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4) 조건에서 배양하였다. 연구 목적에 따라 $TGF-{\beta}1$ (5 ng/ml), Rosiglitazone (50 nM)와 PD98059 ($20{\mu}M$)를 배양액에 첨가하였다. Trypan-Blue와 hematocytometer를 이용하여 현미경하에서 세포의 개수를 측정하였다. Enzyme-linked immunosorbent assay (ELISA)와 western blotting 방법을 사용하여 단백질의 발현 정도를 관찰하였다. 결과 및 결론: 배양액에 $TGF-{\beta}1$을 첨가하여 세포의 증식 정도를 측정한 결과 $TGF-{\beta}1$이 세포의 증식을 억제하는 것을 알 수 있었다. 또한 배양된 세포로부터 PGE2 및 prolactin의 발현을 유도하는 것을 알 수 있었다. 이러한 $TGF-{\beta}1$의 작용은 Smad 및 ERK의 활성화를 통하여 일어남을 알 수 있었다. $PPAR{\gamma}$의 기질인 rosiglitazone을 배양액에 첨가한 결과 $TGF-{\beta}1$에 의한 세포 증식의 억제가 역전되는 것을 알 수 있었다. 뿐만 아니라, 세포 내 ERK의 활성 역시 억제 시켰으며 이 결과 PGE2와 prolactin의 발현이 억제 되는 것을 관찰할 수 있었다. 따라서 본 연구를 통해 $TGF-{\beta}1$에 의한 자궁내막 기질세포의 탈락막화는 Smad와 ERK의 활성화를 통하여 이루어지며 이러한 과정은 $PPAR{\gamma}$에 의해 억제됨을 알 수 있었다. Objective: To investigate the role of ERK and $PPAR{\gamma}$ on the $TGF-{\beta}1$ induced human endometrial stromal cell decidualization in vitro. Method: Endometrial stromal cells are cultured under the following condition: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). $TGF-{\beta}1$ (5 ng/ml), Rosiglitazone (50 nM), and PD98059 ($20{\mu}M$) were added according to experimental purposes. Trypan-Blue and hematocytometer were utilized to count cell number. Enzyme-linked immunosorbent assay (ELISA) and western blotting were utilized to detect proteins. Result: $TGF-{\beta}1$ inhibited proliferation of cultured human endometrial stromal cells and induced expression of PGE2 and prolactin. This effect was mediated by Smad and ERK activation. Administration of rosiglitazone, $PPAR{\gamma}$ agonist, prevented $TGF-{\beta}1$ effect on cell proliferation. Furthermore, Rosiglitazone inhibited $TGF-{\beta}1$ induced activation of ERK, consequently reduced PGE2 and prolactin production. Conclusion: $TGF-{\beta}1$ induced decidualization of endometrial stromal cell through Smad and ERK phosphorylation. $PPAR{\gamma}$ acts as a negative regulator of human ndometrial cell decidualization in vitro.

인간자궁내막의 탈락막화 (Decudualization)에 있어서 TGF-$\beta$ (Transforming Growth Factor-$\beta$)의 역할

박동욱,최동순,김미란,황경주,조미영,안성희,민철기,유희석,Park, Dong-Wook,Choi, Dong-Soon,Kim, Mi-Ran,Hwang, Kyung-Joo,Jo, Mi-Yeong,Ahn, Seong-Hee,Min, Churl-K.,Ryu, Hee-Sug 대한생식의학회 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.1

Objectives: To investigate the role of TGF (Transforming growth factor-$\beta$) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. Design: In the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-$\beta$1 are added. Methods: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining are utilized to detect proteins in the tissue. Results: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-$\beta$1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-$\beta$1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4, 325 pg/ml in E2-dominant and 2, 000 pg/ml in P4-dominant condition compare to 150 pg/ml in no hormone. In separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-$\beta$ and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. Conclusion: It is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-$\beta$ and HGF are two possible paracrine mediators in the human endometrial decidualization.

Differentiation of neural stem cells under co-culture with astrocytes and implantation into the brain of the animal model of Huntington disease

Hwa Lee Ryu(유화리),Taesup Chol(조태섭),Jong Uk Kim(김종욱),Churl K. Min(민철기) 한국생물공학회 2004 한국생물공학회 학술대회 Vol.2004 No.10

새로운 유전자 재조합 방법을 이용한 대장균에서의 인간 tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) 유전자의 가용성 발현

김종욱(Jong Uk Kim),최동순(Dong Soon Choi),주현(Hyun Joo),민철기(Churl K. Min) 한국생물공학회 2008 KSBB Journal Vol.23 No.3

Selective targeting transfection of endometrial cancer cell by cationic polymer against Matrix metalloproteinase (MMP-2)

Joo youn Han(한주연),Dong Soon Choi(최동순),Jin Young Shin(신진영),Hyun Ju Choi(최현주),Churl K. Min(민철기) 한국생물공학회 2004 한국생물공학회 학술대회 Vol.2004 No.10

Peptide Nucleic Acid(PNA)를 이용한 antisense 기법에 적용할 병렬 컴퓨팅용 Bioinformatics tool 개발

김성조(Seongjo Kim),전호상(Hosang Jeon),홍승표(Seungpyo Hong),김현창(Hyon Chang Kim),김한집(Han Jip Kim),민철기(Churl K. Min) 한국정보과학회 2006 한국정보과학회 학술발표논문집 Vol.33 No.1

Unlike RNA interference, whose usage is limited to eukaryotic cells, Peptide Nucleic Acid (PNA) technique is applicable to both eukaryotic and prokaryotic cells. PNA has been proven to be an effective agent for blocking gene expressions and has several advantages over other antisense techniques. Here we developed a parallel computing software that provides the ideal sequences to design PNA oligos to prevent any off-target effects. We applied a new approach in our location-finding algorithm that finds a target gene from the whole genome sequence. Message Passing Interface (MPI) was used to perform parallel computing in order to reduce the calculation time. The software will help biologists design more accurate and effective antisense PNA by minimizing the chance of off-target effects.

ATP 에 의해 유도된 인간 난소 황체화 과립세포의 세포자멸사에 관한 연구

김미란(Mi Ran Kim),박동욱(Dong Wook Park),김영아(Young Ah Kim),조태섭(Tae Seop Cho),황경주(Kyung Joo Hwang),임윤경(Yun Kyoung Lim),민철기(Churl K . Min) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.11

N/A Objective : To determine the effects of extracellular ATP on mitochondrial function and apoptosis during human luteinized granulosa cell cultures. Methods : The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) to luteinized granulosa cells obtained during In vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. Results : Measurement by patch clamp of the granulosa cell membrane potential after ATP addition to cultured granulosa cells showed that both the inward and outward currents were expressed. After treatment of the granulosa cells with JC-1, measurement of the mitochondrial activity by confocal microscopy showed that the with increasing concentrations of ATP the relative ratio of undamaged mitochondria (red/green ratio) tended to decrease (P=0.027). After double staining of the cultured granulosa cells with Annexin V and Propidium Iodide, quantitative flow cytometry analysis showed that apoptosis increased with increasing concentrations of ATP (7.88%, 8.44%, 11.40%, 13.52%, 18.57%). Conclusion : The results of this study shows that apoptosis of granulosa cells increases with increasing extracellular ATP concentrations in cultured human luteinized granulosa cells. This is observed to be a consequence of cell membrane purinoceptor activity and functional changes in the mitochondria. It is therefore thought that remodelling processes of the ovarian tissue is regulated by neuroendocrine factors of the extracellular ATP.

HCG 에 의하여 억제된 ATP 유도 인간 난소 황체화 과립막세포의 세포자멸사에 관한 연구

김영아(Young Ah Kim),김미란(Mi Ran Kim),황경주(Kyung Joo Hwang),박동욱(Dong Wook Park),조태섭(Tae Seop Cho),민철기(Churl K Min),조미영(Mi Yeong Jo) 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.3

N/A Objective: To determine the effects of hCG on extracellular ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) Methods: The addition of various concentrations of ATP (0,0.1,0.25,0.5,0.75mM)and5IuhCG to luteinized granulosa cells obtained during in vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. Results: Calcium imaging with fura-2 revealed that ATP elevated [Ca 2+]i by mobilizing intracellularly stored Ca 2+. A patch clamp study showed that ATP exerted its effect by initially binding to the P2Y type purinoceptor, as evidenced by the ATP-evoked outward Ca 2+ -activated K +current. Probing mitochondria with JC-1, a mitochondrial transmembrane potential-sensitive dye, revealed that ATP induced mitochondrial depolarization in a concentration-dependent manner. A quantitative flow cytometric analysis with Annexin V showed that apoptotic cells were increased in number in proportion to the concentration of ATP, having 18.57% of apoptotic cell populations in the presence of 0.75mM ATP compared to 7.88%in the control. Moreover, treatments with human chorionic gonadotropin (hCG) at 5 IU reversed both the ATP-induced mitochondrial depolarization and apoptosis (18.57 vs 6.32%). Conclusion: Taken together, these results indicate, first, extracellular ATP recognized by the P2Y type purinoceptor on h-LGCs increases the intracellular Ca2+ . Second, the increased intracellular Ca2+ triggers the apoptotic cascade by acting at least, in part, on mitochondria. Third, hCG reverses the ATP-induced apoptosis, raising a possible clinical implication of hCG in the treatment of degeneration of granulosa cells such as follicular atresia.