OPTIMIZATION OF THE TRICHODERMA HARZIANUM CuZn SUPEROXIDE DISMUTASE GENE EXPRESSION IN E. COLI

Main Article Content

Qian Yang*
Li-ming Yang
Pi-Gang Liu
Sen Li
Hu-lun Li

Abstract

A study of the optimization of CuZn superoxide dismutase gene expression in E. coli L21/pET28-SOD was undertaken. The SOD gene was directionally cloned into expression vector pET28.The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG to express SOD protein. The four effective factors, including the initial concentration of BL21/pET28-SOD, concentration of IPTG, incubation temperature and induction time, were optimized during the induction of target protein. To detect the expression efficiency under different induction conditions, the total protein in the samples were subjected to SDS-PAGE. The SOD protein was successfully expressed in E. coli BL21/pET28-SOD. SOD expression was optimal at the following that E. coli BL21/pET28-SOD at OD600nm =0.6, IPTG 0.125 mmol/L, incubation at 37℃ and incubation for four hours. 


Keywords: SOD protein, expression, optimization


Corresponding author: E-mail: YangQ@hit.edu.cn

Article Details

Section
Original Research Articles
Author Biography

Qian Yang*, Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, China

Department of Life Science and Engineering, Harbin Institute of Technology,  Harbin, 150001 P R China 

References

[1] Igor, N. Z., Thoma, J. M., and Rodney, J. F. 2002 Superoxide Dismutase Multigene Family: A Comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) Gene Structures, Evolution, and Expression. Free Radic. Biol. & Med., 3 (33), 337-349.
[2] McCord, J. M., and Fridovich, I. J. 1969 Superoxide dismutase. An Enzymic Function for Erythrocuprein (hemocuprein). Biol. Chem., 244, 6049-6055.
[3] Scandalios, J. G. 1997 Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Plainview, N. Y. 184, 149-161.
[4] Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A., Donalaldson, D., Goto, J., Regan, J. P. and Deng H.x. 1993 Mutations in Cu/Zn Superoxide Dismutase Gene are Associated with Familial Amyotrophic Lateral Sclerosis. Nature, 362, 59-62.
[5] Plantivaux, A., Furla, P., Zoccola, D., Garello, G, Forcioli, D., Richier, S., Merle, P.I., Tambutte, E. and Allemand, D. 2004 Molecular Characterization of two CuZn-superoxide Dismutases in a Sea Anemone, Free Radic Biol. Med., 37(8), 1170-1181.
[6] Hallewell, R. A., Masiarz, F. R., Najarian, R. C., Purna, J. P., Quiroga, M.R., Randolph, A., Sanche, R., Scandella, C. G., Smith, B., Steimer, K. S. and Mullenbach, G. T. 1985 Human Cu/Zu Superoxide Dismutase cDNA: isolation of Clones Synthesising High Levels of Active or Inactive Enzyme from an Expression Library. Nucleic Acids Res., 25(13), 2017-2034.
[7] Hartman, J.R., Geller, T., Yavin, Z., Bartfeld, D., Kanner, D., Aviv, H. and Gorecki, M. 1986 High-level Expression of Enzymatically Active Human Cu/Zn Superoxide Dismutase in Escherichia coli. Proc. Natl. Acad., 83, 7142-7146.
[8] Natvig, D., Imlay, K., Touati, D. and Hallewell, R.A. 1987 Human Copper-zinc Superoxide Dismutase Complements Superoxide Dismutase- deficient Escherichia coli Mutants. J. Biol. Chem., 262, 14697-14701.
[9] Fujii, J., Myint, T., Seo, H.G., Kayanoki, Y., Ikeda, Y. and Taniguchi, N. 1995 Characterization of Wild-type and Amyotrophic Lateral Sclerosirelated Mutant CuZn-superoxide Dismutases Overproduced in Baculovirus-infected Insect Cells. J. Neurochem., 64, 1456-1461.
[10] Kliebenstein, D. J., Monde, R.A., and Last, R. L. 1998 Superoxide Dismutase in Arabidopsis: an Eclectic Enzyme Family with Disparate Regulation and Protein Localization. Plant Physiol., 118, 637-650.
[11] Abarca, D., Roldan, M., Martin, M. and Sabater, B. 2001 Arabidopsis Thaliana Ecotype Cvi Shows an Increased Tolerance to Photo-oxidative Stress and Contains a New Chloroplastic Copper/zinc Superoxide Dismutase Isoenzyme. J. Exp. Bot., 52, 1417-1425.
[12] Bowler, C., Alliotte, T., De Loose, M., Van Montagu, M., and Inze, D. 1989 The Induction of Manganese Superoxide Dismutase in Response to Stress in Nicotiana Plumbaginifolia. EMBO J., 8, 31-38.
[13] Kardish, N., Magal, N., Aviv, D., and Galun, E. 1994 The Tomato Gene for the Chloroplastic CuZn Superoxide Dismutase: Regulation of Expression Imposed in Transgenic Tobacco Plants by a Short Promoter. Plant Mol. Biol., 25, 887-897.
[14] Sakamoto, A., Ohsuga, H., and Tanaka, K. 1992 Nucleotide Sequences of two cDNA Clones Encoding Different Cu/Zn- superoxide Dismutases Expressed in Developing Rice Seed. Plant Mol. Biol., 19, 323-327.
[15] Liu, P. and Yang, Q. 2005 Identification of Genes with a Biocontrol Function in Trichoderma Harzianum Mycelium Using the Expressed Sequence tag Approach. Research in Microbiology, 156, 416-423.
[16] Qu, Z. and Zheng, Y.S. 2000 Cloning, Expression and Purification of N-terminal of Submit PB2 of Influenza Virus RNA Polymerase. Clin. Microb. Immuno., 2(1), 21.