|
|
Cysteine Auxotrophic Expression and Identification of Antiviral Selenium-containing Peptide |
WANG Cheng1,2, SUI Chun-hong1, YAN Gang-lin2, LÜ Shao-wu2, MU Ying2 |
1. School of Basic Medical Sciences, Jilin Medical College, Jilin 132013, China;
2. Key Laboratory of Molecular Enzymology and Engineering, Ministry of Education, College of Life Science, Jilin University, Changchun 130012, China |
|
|
Abstract Objective: To express the soluble antiviral peptide, the cysteine auxotrophic expression system was used in Escherichia coli (E. coli). And the biological activities of the purified antiviral peptide were initially identified. Method: The gene sequence of antiviral peptide Se-GBVA10 was chemically synthesized according to the optimized codons of E.coli, and the gene was cloned into GST fusion expression vector pGEX-2T. Then, the plasmid pGEX-2T-GBVA10 was converted to cysteine auxotrophic expression strains (E. coli BL21 cysE51). Expressed proteins were purified by glutathione Sepharose 4B affinity column. Finally, the content of selenium, the antiviral activity and the antioxidant activity (the vitality of glutathione peroxidase) of the antiviral peptides were detected. Results: The fusion protein, sjGST-Se-GBVA10 and sjGST-GBVA10, were successfully expressed by means of cysteine auxotrophic expression system. After cutting with thrombin, the purified antiviral peptides, Se-GBVA10 and GBVA10 were obtained. The content of selenium of the Se-GBVA10 was 0.974 mol/mol peptide, the glutathione peroxidase vitality of Se-GBVA10 was 47.52 U/μmol, the concentration for 50% of maximal effect (EC50) of Se-GBVA10 was 21.73μmol/L, and the median cytotoxic concentration (CC50) of Se-GBVA10 was 849.41μmol/L. Conclusion: The antiviral peptide Se-GBVA10 has the same antiviral activity as the GBVA10, but also has some antioxidant activity.
|
Received: 08 November 2013
Published: 25 April 2014
|
|
|
|
[1] Mehdi Y, Hornick J L, Istasse L, et al. Selenium in the environment, metabolism and involvement in body functions. Molecules, 2013, 18(3): 3292-3311.
[2] Rayman M P. Selenium and human health. Lancet, 2012, 379(9822):1256-1268.
[3] Schmidt R L, Simonovi ? M. Synthesis and decoding of selenocysteine and human health. Croat Med J, 2012, 53(6):535-550.
[4] Bellinger F P, Raman A V, Reeves M A, et al. Regulation and function of selenoproteins in human disease. Biochem J, 2009, 422(1):11-22.
[5] Liu X, Huang Y, Cheng M, et al. Screening and rational design of hepatitis C virus entry inhibitory peptides derived from GB virus A NS5A. J Virol, 2013,87(3):1649-1657.
[6] Yan F, Yan G, Lv S W, et al. A novel 65-mer peptide imitates the synergism of superoxide dismutase and glutathione peroxidase. Int J Biochem Cell Biol, 2011, 43(12):1802-1811.
[7] Schgger H. Tricine-SDS-PAGE. Nat Protoc, 2006, 1(1): 16-22.
[8] Yin L, Song J, Board PG, et al. Characterization of selenium-containing glutathione transferase zeta1-1 with high GPX activity prepared in eukaryotic cells. J Mol Recognit, 2013, 26(1):38-45.
[9] Battin E E, Brumaghim J L. Antioxidant activity of sulfur and selenium: a review of reactive oxygen species scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms. Cell Biochem Biophys, 2009, 55(1):1-23.
[10] Wang C, Wan P, Gong PS, et al. Novel selenium-containing human single chain variable fragment with glutathione peroxidase activity from computer-aided molecular design. Chem Res Chinese U, 2011, 27(5):813-819.
[11] Jones D M, Gretton S N, McLauchlan J, et al. Mobility analysis of an NS5A-GFP fusion protein in cells actively replicating hepatitis C virus subgenomic RNA. J Gen Virol, 2007, 88(2):470-475.
[12] Bahar I, Timothy R L, Yang L W, et al. Global dynamics of proteins: bridging between structure and function. Annu Rev Biophys, 2010, 39(2): 23-42.
[13] Zwolak I, Zaporowska H. Selenium interactions and toxicity: a review. Cell Biol Toxi, 2012, 28(1) 31-46.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|