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S Yegorova, A Liu, MF Lou; Human Lens Thioredoxin: Cloning, Overexpression, Characterization and H2O2-Upregulation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2365.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Although the cytosolic thioredoxin (TRx-1) gene has been found in human and mouse lenses, this important redox regulating enzyme has never been isolated from the lens. We cloned the TRx-1 gene and purified the recombinant enzyme to study its physiological function in the lens. Methods: Human TRx-1 gene (HTRx-1) was cloned from the human lens cDNA library. The amplified fragments were cloned into pET-15b expression vector and overexpressed in E. coli BL21.The recombinant TRx-1 (RHLTRx-1) was purified by Nickel column followed by SDS-PAGE and Western blot analyses. Thioredoxin reductase (TR) from pig liver was also purified by three-column chromatographic procedures and used for TRx-1 assays. TRx-1 activity was measured by insulin reduction assay while the kinetic studies were done by DTNB reduction assay, both in the presence of NADPH and TR. For upregulating TRx-1, 1.6 million of human lens epithelial cells (HLE-B3) were deprived of serum by incubating with 2% fetal bovine serum overnight and then in serum-free medium for 30 min before subjecting to a bolus of H2O2 (0.1 mM) for 0, 5, 10, 15, 20 and 30 min. The cells were lysed on plate with lysis buffer and used for TRx-1 activity assay and Western blot analysis. Results: The sequence of the lens HTRx-1 gene was identical to that of other human tissues. The purified recombinant enzyme showed a single band of 12 kDa and reacted positively with anti-TRx-1 polyclonal antibody. RHLTRx-1 had a specific activity of 68 U/mg with optimal activity at pH 7.6 and 37ºC. It was sensitive to iodoacetic acid but resistant to oxidation (0.1 mM H2O2) and heat treatment (5 min at 37-85ºC) at a physiological enzyme level (∼10µM). TRx-1 could be upregulated by H2O2 (0.1mM) in cultured HLE-B3 cells, in which Western blot showed 30% increase of TRx-1 protein after 10 min and returned to basal level by 30 min. Conclusion: TRx-1 has been cloned from human lens, expressed in E. coli and purified to homogeneity for the first time. The purified RHLTRx-1 showed similar properties as the TRx-1 from other mammalian tissues.
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