Browsing by Author "Tseng, CF"

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Antioxidant role of human haptoglobin
(2004-08-01)
Tseng, CF
;
Lin, CC
;
Huang, HY
;
Liu, HC
;
Mao, SJT
Human plasma haptoglobin (Hp) is classified according to three phenotypes: Hp 1-1, 2-1, and 2-2 attributed by their two common alleles 1 and 2. Clinically, the 2-2 phenotype is associated with the risk of cardiovascular diseases and diabetes mellitus in patients. In this study, we demonstrate that Hp is an extremely potent antioxidant, which directly protects low density lipoprotein from Cu2+-induced oxidation. Its potency was markedly superior to probucol (one of the most potent antioxidants). Ranking of the IC50 of antioxidant activity was as follows: Hp 1-1 > Hp 2-1 > Hp 2-2 > probucol > vitamin E. Blockage of disulfide linkages between Hp subunits, not only abolished the alpha-helical content but also diminished the ability of Hp to form a complex with hemoglobin. The modified Hp subunits exerted almost 4 times greater antioxidant activity than that of native Hp. To investigate the antioxidant role of Hp on the cellular level, the cDNA of Hp 1-1 was cloned, introduced into the pcDNA3.0 vector which contains the cytomega lovirus promoter and transfected into chinese hamster ovary (CHO)-K1 cells. Following transfection, CHO cells were able to express Hp 1-1 protein and significantly (p < 0.001) elevated cell tolerance against oxidative stress. Transfected cells showed 2-fold higher resistance to hydrogen peroxide exposure for 24 h compared to control cells. Thus, Hp plays a provocative antioxidant role as demonstrated by our in vitro and ex vivo studies.
Expression and purification of human placenta lactogen in Escherichia coli
(2006-04-01)
Lan, PC
;
Tseng, CF
;
Lin, MC
;
Chang, CA
;
生物科技學系
There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quantities of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 degrees C. Determination of the yield of recombinant hPL by SDS-PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8 M urea and purified by a His(6) tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL. (c) 2005 Elsevier Inc. All rights reserved.
Molecular properties of human haptoglobin as probed by monoclonal antibodies
(2004-03-24)
Mao, SJT
;
Yang, YT
;
Lai, SM
;
Wu, CF
;
Chang, TM
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-02-01)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學系
Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp, phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed. (C) 2003 Elsevier Inc. All rights reserved.
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-03-24)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學院
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-02-01)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學系
Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp, phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed. (C) 2003 Elsevier Inc. All rights reserved.