High-level Expression of Human Immune Interferon in Escherichia coli by using tac Promoter

곽규범,현형환,권병세,김광수,Kwack, Kyu-Bum,Hyun, Hyung-Hwan,Kwon, Byung-S.,Kim, Kwang-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3

인간의 면역 인터페론을 코딩하는 유전자를 tac 프로모터와 rrnBT1T2 전사중지서열을 갖는 프라스미드 pKK223-3에 클론닝하여 체세포 유전자에 lacIq 제한차서열을 갖는 대장균 JM105와 체세포 유전자에 lacIq 제한자서열을 갖는 대장균 MM294와 대장균 YMC9에 각각 형질전환시켰다. 생성된 형질전환주를 immunoblotting과 생물학적 역가분석, 전기영동 그리고 전자현미경을 사용하여 확인했다. 면역 인터페론의 발현은 대장균 JM105에서 효율적으로 조절되었으며, 대장균 MM294와 대장균 YMC9에서는 IPTG의 처리없이도 생산됨을 발견하였다. 또한 면역 인터페론이 용해된 형태와 불용성 상태인 inclusion body로 생성됨을 알았다. 이때 생산성은 $9.7{\times}10^9IU/l$이며 면역 인터페론 단백질이 대장균의 전체 단백질중 약 30%에 해당함을 알았다. The cDNA for the structural sequence of mature human mmune-interferon ($IFN-{\gamma}$) was introduced into E. coli high expression vector, pKK223-3, constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. $IFN-{\gamma}$, which cross-reacted with mouse anti human $IFN-{\gamma}$ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coii MM294, and E. coli YMC9 by using the IPTG.

대장균에서 인간의 면역 인터페론을 tac 프로모터를 이용하여 고도로 발현시키는 연구

곽규범,현형환,권병세,김광수 ( Kyu Bum Kwack,Hyung Hwan Hyun,Byung S . Kwon,Kwang Soo Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

The cDNA for the structural sequence of mature human mmune-interferon (IFN-γ) was introduced into E. coli high expression vector, pKK223-3,constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. IFN-γ, which cross-reacted with mouse anti human IFN-γ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coli MM294, and E. coli YMC9 by using the IPTG.

Interaction Effects of Lipoprotein Lipase Polymorphisms with Lifestyle on Lipid Levels in a Korean Population: A Cross-sectional Study

편정아,곽규범,김선신,박경채,백인경,조남희,고인송,이종영,조윤신,김영진,고민진,심유진,신철 한국유전체학회 2012 Genomics & informatics Vol.10 No.2

Lipoprotein lipase (LPL) plays an essential role in the regulation of high-density lipoprotein cholesterol (HDLC) and triglyceride levels, which have been closely associated with cardiovascular diseases. Genetic studies in European have shown that LPL single-nucleotide polymorphisms (SNPs) are strongly associated with lipid levels. However, studies about the influence of interactions between LPL SNPs and lifestyle factors have not been sufficiently performed. Here, we examine if LPL polymorphisms, as well as their interaction with lifestyle factors, influence lipid concentrations in a Korean population. A two-stage association study was performed using genotype data for SNPs on the LPL gene, including the 3′ flanking region from 7,536 (stage 1) and 3,703 (stage 2) individuals. The association study showed that 15 SNPs and 4 haplotypes were strongly associated with HDLC (lowest p = 2.86 × 10−22) and triglyceride levels (lowest p = 3.0 × 10−15). Interactions between LPL polymorphisms and lifestyle factors (lowest p = 9.6 × 10−4) were also observed on lipid concentrations. These findings suggest that there are interaction effects of LPL polymorphisms with lifestyle variables, including energy intake, fat intake, smoking, and alcohol consumption, as well as effects of LPL polymorphisms themselves, on lipid concentrations in a Korean population.

다관절 로봇 암 기반 고속 열 성형 공정을 활용한 열가소성 복합재 부품 평가

신호영,노지섭,박규범,석창민,권진회,곽병수,남영우,Ho-Young Shin,Ji-Sub Noh,Gyu-Beom Park,Chang-Min Seok,Jin-Hwe Kweon,Byeong-Su Kwak,Young-Woo Nam 한국복합재료학회 2023 Composites research Vol.36 No.5

본 연구에서는 다관절 로봇 암을 활용한 열 성형 공정을 통해 열가소성 복합재 부품을 개발하였다. 유한요소해석을 기반으로 로봇 암이 적용된 고속 열 성형 공정의 최적 공정 변수를 도출하였고, 이를 기반으로 실제 제작 공정을 통해 공정 변수를 구체화하였다. 제작된 부품과 유한요소해석 간의 두께 균일성, 주름 분포를 비교하였고, 이를 통해 유한요소해석의 타당성을 입증하였다. 또한, 제작된 복합재 부품의 성형성을 평가하기 위해 결정화도와 기공률을 측정하였고, 그 결과 우수한 성형성을 보인 것으로 평가하였다. 이에 따라 본 연구는 로봇 암기반의 고속 열 성형 공정을 통한 공정 확립과 이를 통한 복합재 구조의 제작 가능성을 확인하였다.

Epistasis between SNPs in genes involved in lipoprotein metabolism influences high- and low-density lipoprotein cholesterol levels

김선신,고정재,곽규범,신철,조남한,고인송 한국유전학회 2014 Genes & Genomics Vol.36 No.6

Although genome-wide association (GWA)studies have provided valuable insights into the geneticarchitecture of human disease, they have elucidated relativelylittle of the heritability of complex traits. A significantpart of the missing heritability might be explained byrare combinations of common SNPs. We hypothesized thatepistasis among 15 genes (148 SNPs) involved in lipoproteinmetabolism would influence HDL-cholesterol(HDL-C) and LDL-cholesterol (LDL-C) levels. UsingSNPwinter software with the various epistatic models, weidentified 58 association signals with HDL-C levels forSNPs in eleven genes and 118 associations with LDL-C forSNPs in fourteen genes. These associations were discoveredin the urban Ansan cohort (n = 4,102) and replicatedin a rural cohort (n = 3,434), the Ansung. We found replicatedassociations with new genes (SOAT1, APOB,HMGCR, and FDFT1 for HDL-C, and SOAT1, FDFT1,LPL, SQLE, ABCA1, LRP1, SCARB1, and PLTP for LDLC),in addition to those (CETP, LIPC, LPL, ABCA1, PLTP,SCARB1, and LRP1 for HDL-C, and CETP, LIPC, LDLR,APOB, CYP7A1, and HMGCR for LDL-C) identified byGWA studies, through investigating pairwise interactionsbetween candidate genes of biological and clinical importance. Interestingly, we found that some genes were morelikely to be involved in epistatic interactions (ABCA1 andLIPC for HDL-C, and ABCA1, SCARB1, and LIPC forLDL-C).

사람 중간엽줄기세포 성장에 미치는 basic fibroblast growth factor의 영항

김성수,최정원,곽규범,이영돈,서해영 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.6

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established conditions that allowed a long term expansion of hMSCs. To search for the optimum culture condition, growth rates of hMSCs were measured in the presence of several growth factors. Hepatic growth factor (HGF) and leukemia inhibitory factor (LIF) did not facilitate proliferation of hMSCs. In contrast, basic fibroblast growth factor (bFGF) effectively promoted growth of the cells in vitro by 3 fold. The growth stimulatory effect of bFGF was dependent on the concentration. The adipogenic potential was dramatically decreased in hMSCs isolated from an aged donor whereas osteogenic potential was minimally decreased. Addition of bFGF resumed the adipogenic and osteogenic differentiation potential. Thus, the cells that expanded in the presence of bFGF retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineage cells. MSCs could be expanded for at least 8 passages with bFGF and the resulting cells retained the normal karyotype. The cells were positive for CD9, CD13, CD15, CD90, CD137, and CD140b; but negative for CD14, CD34, and CD45. Importantly, the cells were found to express a neural stem cell marker, nestin, and a neuronal marker, β-tubulin III. The results suggest that bFGF promote proliferation while maintaining multi-lineage differentiation potency of hMSCs. Finally, we suggest that it is critical to identify novel markers other than nestin or β-tubulin III to monitor acquisition of neuronal phenotypes by hMSCs. 중간엽줄기세포는 중배엽유래의 세포로 분화할 수 있는 다분화능을 갖는 줄기세포로서 골수에서 쉽게 채취할 수 있다. 본 연구에서는 사람의 중간엽줄기세포를 시험관내에서 장기간 배양시 세포의 증식과 다분화능에 있어서 basic fibroblast growth factor (bFGF)의 영향을 조사하였다.세포를 배양할 수 있는 최적의 조건을 설정하기 위하여 여러 성장인자를 시험하였다. Hepatic growth factor (HGF)와 leukemia inhibitory factor (LIF)는 골수에서 분리한 중간엽줄기세포의 증식에 별다른 영향을 주지 않았으나, bFGF는 시험관내에서 세포의 성장을 3배정도 증가시켰으며, 그 효과에 있어서 bFGF의 농도가 10 ng/ml 이상인 경우에는 큰 차이가 없었다. 저연령층의 골수에서 추출한 중간엽줄기세포는 bFGF의 존재와 상관없이 지방세포, 뼈세포, 연골세포로 분화하는 잠재력을 유지하고 있었으며 CD9, CD13, CD15, CD90, CD137, CD140b에 양성이었고, CD14, CD34, CD45에는 음성이었으며, 정상적인 핵형을 유지하고 있었다. 반면에 고령의 공여자로부터 분리한 중간엽줄기세포는 bFGF가 없을 경우 지방세포로의 분화능이 현격히 낮았으나, bFGF가 있는 경우에는 지방세포 및 뼈세포로의 분화능이 유지되었다. 또한 bFGF가 첨가된 배양액에서 증식시킨 중간엽줄기세포는 미분화상태에서도 이미 신경줄기세포의 표지자인 nestin과 신경세포의 표지자인 β-tubulin III을 발현하고 있었다. 이러한 결과는 bFGF가 사람의 중간엽줄기세포의 증식과 다분화능을 유지시키는데 중요한 성장인자로 작용함을 의미하고 있다. 또한, 중간엽줄기세포를 신경계질환의 세포치료제로서 사용하기 위해서는 미분화상태와 신경세포로 분화된 후를 구별할 수 있는 새로운 표식인자가 필요함을 제시하고 있다.

(1,3)$\beta$-Glucansynthase효소 억제 활성을 가진 천연물의 검색

천현자,김영순,이영행,곽규범,권석용,권태오,채규윤,Chun Hyun Ja,Kim Young Sun,Lee Young Hang,Kwak Geu Byum,kwon Suk young,Kwon Tae Oh,Chai Geu Yun 대한동의생리학회 2003 동의생리병리학회지 Vol.17 No.6

Antifungal activities of the extracts from 26 medicinal plants were investigated utilizing paper-disk diffusion method and (1,3)β-glucan synthase inhibitory assay. (1,3)β-glucan synthase is considered as valuable target in the development of antifungal agents. Among the screened extracts, the ethyl acetate extract of Equisetum arvense, the ethyl acetate extract of Polygonum aviculare, the butanol extract of Crataegus pinnatifida and the n-hexane extract of Saussurea lappa showed significant antifungal activities on Candida albacans in both disk diffusion and enzyme assays.

유전자 조작 알파 인터페론의 조직분포에 관한 연구

김제학,이혜선,김달현,조남진,곽규범 한국약제학회 1987 Journal of Pharmaceutical Investigation Vol.17 No.4

The distribution features of recombinant human alpha-interferon (rHuIFN- αA) and ^(14)C-radiolabeled rHuIFN- αA (^(14)C-rHuIFN- αA) were investigated in ICR mice after i.v. injection. The level of rHuIFN- αA in the kidney was significantly higher than those in lung and liver at 10min after the injection. But the level was reduced significantly at 60min. The level of radioactivity in the kidney was also significantly higher than those in other organs after i.v. injection of ^(14)C-rHuIFN- αA, but it was reduced at much slower speed than was rHuIFN- αA. These results show that interferon is distributed repidly and the kidney is the main site of distribution and metabolism of rHuIFM- αA.

Screening study for genetic polymorphisms affecting pharmacokinetics of pioglitazone

윤지영,김보형,이지현,이기동,곽규범,임성빈 대한임상약리학회 2016 Translational and Clinical Pharmacology Vol.24 No.4

Pioglitazone is known to have antidiabetic effects through decreasing peripheral, hepatic and vas¬cular insulin resistance by the stimulation of PPAR gamma. To address the possible genetic factors affecting the pharmacokinetics (PK) of pioglitazone, 27 male Korean volunteers were enrolled from two separate bioequivalence studies. Each subject was administered 15 mg pioglitazone and reference drug PK parameters were used. We used Illumina Human610 Quad v1.0 DNA Analysis BeadChip for whole genome SNPs analysis and whole genome genotyping data was processed by linear regression analysis for PK parameters. We found 35 significant SNPs (P < 0.0001) in Cmax, 1,118 significant SNPs (P < 0.0001) in Tmax and 1,259 significant SNPs (P < 0.0001) in AUCinf from whole genome analysis. For clinical pharmacological purpose, we selected SNPs from several phase I and II drug metabolizing enzyme and analyzed PK parameters with genotypes. Four SNPs (rs7761731 and rs3799872 from CYP39A1; rs156697 from GSTO2; rs1558139 from CYP4F2) showed significant associations with pioglitazone Cmax. In the Tmax group, seven SNPs from 3 genes (rs3766198 from CYP4B1; rs2270422 from GSTZ1; rs2054675, rs10500282, rs3745274, rs8192719, and rs11673270 from CYP2B6) had significant associations. In the AUCinf group, seven SNPs from 4 genes (rs11572204 from CYP2J2; rs4148280 from UGT2A1, rs4646422 from CYP1A1; rs3745274, rs8192719, rs11673270, and rs707265 from CYP2B6) showed significant associations with piogli¬tazone absorption. These results showed that genetic makeup could affect the PK parameters and these informations could be provide information for personalized pioglitazone therapy.

Screening study for genetic polymorphisms affecting pharmacokinetics of simvastatin

임소희,김보형,이기동,곽규범,임성빈 대한임상약리학회 2016 Translational and Clinical Pharmacology Vol.24 No.1

Simvastatin reduces plasma cholesterol by inhibiting HMG-CoA reductase (HMGR) and is widely used in the treatment of hypercholesterolemia. To screening the possible genetic factors affecting the pharmacokinetics (PK) of simvastatin, 35 male Korean volunteers were enrolled from two sepa¬rate bioequivalence studies. Each subject was administered 20 mg simvastatin and reference drug PK parameters were used. We used Illumina Human610Quad v1.0 DNA Analysis BeadChip for whole genome SNPs analysis and whole genome genotyping data was processed by linear regression analysis for PK parameters of drug metabolizing enzymes and transporters. We found 145 signifi¬cant SNPs (P < 0.01) in Cmax, 135 significant SNPs (P < 0.01) in Tmax and 85 significant SNPs (P < 0.01) in AUCinf from whole genome analysis. In particular, we found that the ABCC2 gene had a significant effect on Cmax and AUCinf. These results could provide information of possible candidate genes for personalized simvastatin therapy.