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| Expression, Purification and Enzymatic Characteristics of Human Hex D in E.coli |
| LIU Lin1, XU Yan2, CAI Chun-mei3, LI Jing2, CAI Yu-mei 1 |
1. College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, China;
2. College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China;
3. Dezhou Second People’s Hospital, Dezhou 253024, China |
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Abstract Glycosylation is an important protein post-translational modification, which is involved in many cellular processes, including signal transduction and cell recognition. Properly hydrolysis of glycoconjugates is essential for organism metabolism. Human hexosaminidase D (Hex D) is a newly discovered glycosidase which cleaves GalNAc ( O-linked N-Acetyl-β-D-glatosamine) modification. However, the enzymatic characteristics of Hex D remains unknown. Here, the cDNA sequence was amplified by PCR and cloned into plasmid pET3C. After transformed the recombinant plasmids into Escherichia coli BL21 (DE3) plys, the expression of Hex D was optimized with 0.1 mmol/L IPTG in 10 hours and purified it with the Ni-NTA affinity chromatography. SDS-PAGE verified the molecular weight (58 kDa) and the purity (> 95%). Using 4-MU-GalNAc (4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-galctopyranoside)as substrate, the optimal pH and temperature for Hex D is 5.5 and 37℃ respectively. Assay of Hex D pre-incubated for 30 min at different temperature indicated that it had high thermal stability and retain activity at 50℃. 1mmol/L metal ions(CuSO4、FeSO4·7H2O、MgCl2·6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)and EDTA has no different effect on Hex D enzymic activity. However,10mmol/L AlCl3,CuSO4 or FeSO4·7H2O decreased the activity of Hex D to different extent. Using the optimum pH and temperature the Km value and Vmax of Hex D were 0.16mmol/L and 3.06 μmol/(min·mg) respectively.
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Received: 27 April 2012
Published: 25 September 2012
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