• BMB Reports
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• 제31권1호
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• pp.77-82
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• 1998

To purify the geranylgeranyl protein transferase type I (GGPT-I) efficiently, a gene expression system using the pGEX-4T-1 vector was constructed. The cal1 gene, encoding the ${\beta}$ subunit of GGPT-I, was subcloned into the pGEX-4T-1 vector and co-transformed into E. coli cells harboring the ram2 gene, the ${\alpha}$ subunit gene of GGPT-I. GGPT-I was highly expressed as a fusion protein with glutathione S-transferase (GST) in E. coli, purified to homogeneity by glutathione-agarose affinity chromatography, and the GST moiety was excised by thrombin treatment. The purified yeast GGPT-I showed a dose-dependent increase in the transferase activity, and its apparent $K_m$ value for an undecapeptide fused with GST (GST-PEP) was $0.66\;{\mu}M$ and the apparent value for geranylgeranyl pyrophosphate (GGPP) was $0.071\;{\mu}M$.

• BMB Reports
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• 제31권3호
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• Parasites, Hosts and Diseases
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• 제39권3호
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• pp.241-246
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• 2001

The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins. The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T: OFR without signal sequence and C-terminal hydrophobic sequence, S: N-terminal 2/3 parts of S, A: C-terminal 2/3 parts, P; N-terminal 1/3 part, X: middle 1/3 part Y; and C-terminal 1/3 part, Z, respectively. The DNA fragments were cloned into pGEX-47 vector. Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T,66 kDa for S, 52 kDa for A,53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE. The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot. These findings suggest that the antigenic domain of Nc-p43 of N. caninum may be localized in the C-terminal 2/3 parts. Together with Al9 clone in SAGI of Toxoplasma gondii (Nam et at., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species .

• Parasites, Hosts and Diseases
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• 제51권5호
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• pp.503-510
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• 2013

Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.

• Journal of Nutrition and Health
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• 제44권5호
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• pp.366-377
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• 2011

Glutathione S-transferase (GST) is a multigene family of phase II detoxifying enzymes that metabolize a wide range of exogenous and endogenous electrophilic compounds. GSTM1 and GSTT1 gene polymorphisms may account for inter-individual variability in coping with oxidative stress. We investigated the relationships between the level of lymphocyte DNA and antioxidative parameters and the effect on GST genotypes. GSTM1 and GSTT1 were characterized in 301 young healthy Korean adults and compared with oxidative stress parameters such as the level of lymphocyte DNA, plasma antioxidant vitamins, and erythrocyte antioxidant enzymes in smokers and non smokers. GST genotype, degree of DNA damage in lymphocytes, erythrocyte activities of superoxide dismutase, catalase, and glutathione peroxidase (GSH-Px), and plasma concentrations of total radical-trapping antioxidant potential (TRAP), vitamin C, ${\alpha}$- and ${\gamma}$-tocopherol, ${\alpha}$- and ${\beta}$-carotene, and cryptoxanthin were analyzed. Lymphocyte DNA damage assessed by the comet assay was higher in smokers than that in non-smokers, but the levels of plasma vitamin C, ${\beta}$-carotene, TRAP, erythrocyte catalase, and GSH-Px were lower than those of non-smokers (p < 0.05). Lymphocyte DNA damage was higher in subjects with the GSTM1- or GSTT1-present genotype than those with the GSTM1-present or GSTT1- genotype. No difference in erythrocyte antioxidant enzyme activities, plasma TRAP, or vitamin levels was observed in subjects with the GSTM1 or GSTT1 genotypes, except ${\beta}$-carotene. Significant negative correlations were observed between lymphocyte DNA damage and plasma levels of TRAP and erythrocyte activities of catalase and GSH-Px after adjusting for smoking pack-years. Negative correlations were observed between plasma vitamin C and lymphocyte DNA damage only in individuals with the GSTM1-present or GSTT1- genotype. The interesting finding was the significant positive correlations between lymphocyte DNA damage and plasma levels of ${\alpha}$-carotene, ${\beta}$-carotene, and cryptoxanthin. In conclusion, the GSTM1- and GSTT1-present genotypes as well as smoking aggravated antioxidant status through lymphocyte DNA damage. This finding confirms that GST polymorphisms could be important determinants of antioxidant status in young smoking and non-smoking adults. Consequently, the protective effect of supplemental antioxidants on DNA damage in individuals carrying the GSTM1- or GSTT1-present genotypes might show significantly higher values than expected.

• BMB Reports
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• 제31권4호
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• pp.399-404
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• 1998

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the $I_{50}$ values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.

• 대한전기학회:학술대회논문집
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• 대한전기학회 2006년도 제37회 하계학술대회 논문집 C
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• pp.1438-1439
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• 2006

$Ge_{2}Sb_{2}Te_5$(GST) thin film at present is a promising candidate for a phase change random access memory (PCRAM) based on the difference in resistivity between the crystalline and amorphous phase. PCRAM is an easy to manufacture, low cost storage technology with a high storage density. Therefore today several major chip in manufacturers are investigating this data storage technique. Recently, A. Pirovano et al. showed that PCRAM can be safely scaled down to the 65 nm technology node. G. T Jeonget al. suggested that physical limit of PRAM scaling will be around 10 nm node. Etching process of GST thin ra films below 100 nm range becomes more challenging. However, not much information is available in this area. In this work, we report on a parametric study of ICP etching of GST thin films in $Cl_2$/Ar chemistry. The etching characteristics of $Ge_{2}Sb_{2}Te_5$ thin films were investigated using an inductively coupled plasma (ICP) of $Cl_2$/Ar gas mixture. The etch rate of the GST films increased with increasing $Cl_2$ flow rate, source and bias powers, and pressure. The selectivity of GST over the $SiO_2$ films was higher than 10:1. X-ray photoelectron spectroscopy(XPS) was performed to examine the chemical species present in the etched surface of GST thin films. XPS results showed that the etch rate-determining element among the Ge, Sb, and Te was Te in the $Cl_2$/Ar plasma.

• Journal of Nutrition and Health
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• 제50권1호
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• pp.10-24
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• 2017

본 연구는 건강한 성인 남녀를 대상으로 glutathione S-transferase (GST)M1 및 T1 유전자 다형성에 따라 한식에서 주로 섭취하는 식물성 식품군과 한식 식단의 DNA 손상 감소효과를 측정하여 유전적 민감도가 어떻게 나타나는지를 알아보기 위해 수행되었다. 이를 위하여 건강한 성인 남녀 59명을 대상으로 혈액을 채취하여 GST genotype을 분류하였으며 그 중 17명을 선발하여 DNA 손상 감소효과를 Comet assay를 이용하여 측정하였고 DNA damage relative score로 나타냈다. 제 5기 2차년도 국민건강영양조사를 활용하여 한국인이 많이 섭취하는 식물성 식품을 10가지 식품군 (감자류, 견과류, 곡류, 과일류, 김치류, 두류, 버섯류, 오일류 채소류 해조류)으로 분류 후, 각 식품군별 총 섭취량의 1% 이상을 섭취한 84종의 식품을 한식 식물성 식품으로 최종 선정하였으며 한식 식단 (Korean diet)은 한국영양학회에서 발행한 [2010 한국인 영양섭취 기준]에 제시되어 있는 1주일 표준식단 (2,000 kcal/day)을 사용하였다. GSTM1 유전자 다형성에 따른 한식 식물성 식품군의 Tail moment로 본 DNA 손상 감소효과는 곡류와 오일류에서만 GSTM1 wild type보다 mutant type에서 유의하게 높았다. 이에 비해 DNA 손상 감소 효과를 % DNA in tail과 Tail moment로 본 결과, 견과류 과일류 채소류 버섯류 김치류 해조류에서 GSTT1 mutant type에 비해 wild type에서 유의하게 더 높게 나타났다. GSTM1과 GSTT1의 combined genotype에 따라 한식 식물성 식품의 DNA 손상 감소효과를 본 결과, 과일류, 김치류, 버섯류, 채소류, 해조류는 1군 (GSTM1+/GSTT1+) 및 3군 (GSTM1-/GSTT1+)에서, 오일류는 3군 (GSTM1-/GSTT1+)에서 DNA 손상 감소 효과가 유의하게 높았으며. 감자류, 견과류, 곡류, 두류, Total은 DNA 손상 감소 효과가 2군 (GSTM1+/GSTT1-) 및 3군 (GSTM1-/GSTT1+)에서 유의하게 높아 식품군에 따라 GST 유전자 다형성에 따른 DNA 손상 감소효과가 다르게 나타나는 것을 확인할 수 있었다. 한식 식단은 DNA 손상의 세 가지 지표인 % DNA in tail, Tail moment, Tail length로 측정해본 결과 GSTM1의 경우 wild type에서 mutant type보다 더 크게 나타났으며, GSTT1의 경우는 genotype에 따라 DNA 손상이 달라지는 경향은 있었지만 유의한 차이를 나타내지 않았다. 결론적으로 한식에서 주로 섭취하는 식물성 식품군에서는 식품에 따라 부분적으로 GSTM1은 mutant type에서, GSTT1은 wild type에서 DNA 손상 보호효과가 더 크게 나타났으며, GSTM1과 GSTT1의 combined genotype에 따른 DNA 손상 보호효과는 식품군에 따라 다르게 나타났다. 반면, 한식 식단에서는 DNA 손상 보호효과가 GSTM1 wild type에서 mutant type보다 더 크게 나타났으며, GSTT1 genotype에는 영향을 받지 않는 것으로 나타났다. 이와 같은 결과는 한식 식물성 식품군 및 식사패턴의 항산화 기능 우수성을 증명하는 기초자료가 될 것이며, 나아가 개인별 유전자에 따른 항산화 맞춤영양연구를 시작하는 시발점이 될 수 있을 것이다. 앞으로 GST 유전자 다형성에 따른 한식과 한식 식물성 식품군의 유전적 민감도를 더 명확하게 규명하기 위해서는 대상 인원을 늘려 수행하는 광범위한 연구가 필요할 것으로 보인다.

• BMB Reports
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• 제29권5호
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• pp.462-467
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• 1996

Acetolactate synthase (ALS, EC 4.1.3.18) is the first common enzyme in the biosynthesis of leucine, isoleucine, and valine. It is the target enzyme for several classes of herbicides, including the sulfonylureas, the imidazolinones, the mazolopyrimidines, the pyrimidyl-oxy-benzoates, the pyrimidyl-thio-benzens, and the 4,6-dimethoxypyrimidines. An amino-terminal fragment of the sulfonylurea-resistant ALS gene (SurB) from Nicotiana tabaccum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS1 was used to transform Escherichia coli strain BL21, and the tobacco ALS was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). Polyclonal antibodies against the fusion product (GST-ALS) were produced, and the sensitivity of GST-ALS with the rabbit anti-GST-ALS IgG was up to 50 ng. This antibody was used for Western blot analysis of the partially purified ALS from barley shoots. The results suggest that the polyclonal antibody produced in this study can be used to detect plant ALS.

• Asian Pacific Journal of Cancer Prevention
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• 제17권4호
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• pp.2145-2150
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• 2016

Background: Infection with hepatitis B virus (HBV) is a major global public health problem, with a wide spectrum of clinical manifestations. Human cytosolic glutathione-S-transferases (GSTs) include several classes such as alpha (A), mu (M), pi (P), sigma (S), zeta (Z), omega (O) and theta (T). The present study aimed to investigate the role of GST omega genes (GSTO1 and GSTO2) in different groups of patients infected with HBV. Materials and Methods: HBV groups were classified according to clinical history, serological tests and histological analysis into normal carriers (N), acute (A), chronic (CH), cirrhosis (CI) and hepatocellular carcinoma (HCC) cases. The study focused on determination of the genotypes of GST omega genes (GSTO1 and GSTO2) and GST activity and liver function tests. Results: The results showed that GSTO1 (A/A) was decreased in N, A, CH, CI and HCC groups compared to the C-group, while, GSTO1 (C/A) and GSTO1(C/C) genotypes were increased significantly in N, A, CH, CI and HCC groups. GSTO2 (A/A) was decreased in all studied groups as compared to the C-group but GSTO2(A/G) and GSTO2(G/G) genotypes were increased significantly. In addition, GST activities, albumin and TP levels were decreased in all studied groups compared to the C-group, while the activities of transaminases were increased to differing degrees. Conclusions: The results indicate that GSTO genetic polymorphisms may be considered as biomarkers for determining and predicting the progression of HBV infection.