title: Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum.
abstract: BACKGROUND
  CuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research.
  
  
  RESULTS
  High-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L(-1) of culture volume and the bacterial biomass was ~ 4.5 g L(-1). The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage.
  
  
  CONCLUSIONS
  Overexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India. narendra@icgeb.res.in.
authors.forename: Narendra
authors.initials: N
authors.lastname: Tuteja
authors.affiliation: Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India. panchanand51@gmail.com.
authors.forename: Panchanand
authors.initials: P
authors.lastname: Mishra
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India. sandeep@icgeb.res.in.
authors.forename: Sandep
authors.initials: S
authors.lastname: Yadav
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India. marjant1359@yahoo.com.
authors.forename: Marjan
authors.initials: M
authors.lastname: Tajrishi
authors.affiliation: Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India. sudhir.baral88@gmail.com.
authors.forename: Sudhir
authors.initials: S
authors.lastname: Baral
authors.affiliation: Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India. surendrachandra@gmail.com.
authors.forename: Surendra Chandra
authors.initials: SC
authors.lastname: Sabat
mesh_terms: D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D018532:Pisum sativum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013862:Thiogalactosides / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013862:Thiogalactosides; D013482:Superoxide Dismutase