Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris (original) (raw)
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Objective Superoxide dismutase (SOD) enzyme has implications in modulating the cell's redox state. The study aims to explore the host genetic factors that limit the heterologous expression of a thermostable SOD from Potentilla atrosanguinea (Pa-SOD) in E. coli. Results It was observed that the heterologous expression of Pa-SOD in E. coli did not exhibit any enhancement after 1 h of induction. This led to the alteration in cell morphology and an increase in the doubling time of E. coli cells expressing Pa-SOD. Label-free quantification and MALDI-TOF/TOF-MS/ MS analysis suggested differential expression of 81 proteins, of which 77 proteins were found to be downregulated and 4 were found to be upregulated in Pa-SOD expressing cells as compared to uninduced E. coli cells. Functional analysis of downregulated proteins shows involvement in molecular function, biological process, and were the part of a cellular component. The STRING database revealed interaction of an essential autoregulatory protein, RNase E with other proteins involved in biosynthetic processes, protein biosynthesis and folding, and cell division. Further, validation of RNase E protein revealed upregulation of rne at transcript level and downregulation of RNase E at protein level as compared to uninduced cells. Conclusions The observations suggested the operation of multifaceted mechanisms with a key role of RNase E that regulated the expression of Pa-SOD at the physiological and molecular level. Since Pa-SOD has commercial applications, identification and manipulation of these networked genetic factors could lead to improvement of host strain for large-scale production of biologically active Pa-SOD and other heterologous proteins.
Process Biochemistry, 2009
Superoxide dismutase (SOD) catalyzes the conversion of the superoxide radical ( O 2 À ) into oxygen and hydrogen peroxide. Deschampsia antarctica is a plant that grows in Antarctica and survives to extreme low temperature and high UV radiation, thus it is an ideal model to study novel antioxidants. A cDNA Cu/Zn-SOD gene from D. antarctica was cloned into a pET vector and expressed in Escherichia coli BL21-SI. 112 mg/L of recombinant Cu/Zn-SOD was attained in batch cultures in bioreactor. Using Ni-affinity gel chromatography, the recombinant Cu/Zn-SOD was recovered with a purity of 90% and a specific enzyme activity of 749 at 25 8C. However, zymogram test showed that the enzyme has more activity at 4 8C. This D. antarctica SOD could be used to reduce the oxidation of refrigerated and frozen foods. ß
Purification and partial characterization of Cu/Zn superoxide dismutase from
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996 U mg of protein(-1)) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34,034 Da and has high N-terminal homology with other yeasts' Cu/Zn SOD enzymes. The protein is characterized with some unique features such as-thermostability (t(1/2) at 70 degrees C=30 min), pH stability in the alkaline range (7.5-8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme.
Biotechnology Letters, 2000
A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg−1) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K d) of the enzyme was 2.46 × 10−3 min−1 and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.
Febs Letters, 1995
We have purified the Cu,Zn superoxide dismutase (CuZnSOD) from the periplasmic space of an Escherichia coli strain unable to synthesize MnSOD and FeSOD. Gel filtration chromatography evidenced that under all the experimental conditions tested the enzyme was monomeric. The catalytic activity of this CuZnSOD was comparable to that of other well characterized dimeric eukaryotic isoenzymes, indicating that a dimeric structure is not essential to ensure enzymatic efficiency. Furthermore the purified enzyme proved to be highly heat-stable and, uniquely among CuZnSODs, protease-sensitive. The latter property may explain the previously described lability of this protein in cell extracts.
Archives of Biochemistry and Biophysics, 1997
Brucella abortus (8) and other Brucella species (9), The periplasmic Cu,Zn superoxide dismutase Haemophilus influenzae, H. parainfluenzae (10), and (Cu,ZnSOD) from Escherichia coli has been shown other Haemophilus species (11). It began to appear by sedimentation equilibrium to be a monomer with likely that Cu,ZnSODs were in fact widely distributed a molecular weight of Ç17,000. The enzyme suffered among gram-negative bacteria and this change of view a reversible inactivation when heated to 70ЊC. This gained impetus from the finding of Cu,ZnSOD in Eschewas minimized by added Cu(II) or Zn(II). Heat labilrichia coli (12), in Salmonella typhimurium (13), and ity was greater in phosphate than in Tris buffer. The in Legionella pneumophila (14). enzyme exhibited a time-dependent inactivation by In P. leiognathi (15), C. crescentis (16, 17), E. coli (12, Hg(II) and this too was greater in phosphate than 18), S. typhimurium (13), B. abortus (19), and in L. in Tris. This behavior can be explained by a modest pneumophila (20), the Cu,ZnSOD is periplasmic. It is affinity of the enzyme for Cu(II) and Zn(II) which not yet certain whether there are periplasmic sources results in a dissociation/association equilibrium. Elof O 0 2 , or whether this Cu,ZnSOD protects primarily evation of the temperature shifts this equilibrium against exogenous O 0 2. However, it is clear that this toward dissociation and phosphate sequesters the enzyme is induced late in the growth cycle (12, 21, 22), released metals making them less available for reinis important for survival in stationary phase (14), and, sertion at the active site. Hg(II) competes for occuin mutants lacking SodA and SodB, contributes to the pancy of the active site and there were more unoccuability to grow aerobically (23). Whether the Cu,ZnSOD pied sites in phosphate than in Tris. A parallel was functions as a pathogenicity factor in B. abortus has drawn between the E. coli Cu,ZnSOD and FALS vabeen suggested but is still unsettled (24, 25). rients of human Cu,ZnSOD, which are also relatively When first studied (12) the E. coli Cu,ZnSOD was unstable and exhibit low affinity for Cu(II). ᭧ 1997 found to be much less stable than the more thoroughly Academic Press characterized eukaryotic Cu,ZnSODs and, like them, was assumed to be dimeric. The subsequent report of a stable, monomeric Cu,ZnSOD from E. coli (26) indi-Copper-and zinc-containing superoxide dismucated that the stability and the quaternary structure tases (Cu,ZnSODs) 3 were thought to be restricted to of the E. coli Cu,ZnSOD would merit further study. We eukaryotes (1-4). This conventional wisdom was therefore used sedimentation equilibrium to establish called into question by sporadic reports of Cu,ZnSOD the molecular weight of this enzyme and examined in diverse bacteria. The first was from the marine those factors which influence its stability. The results bacterium Photobacter leiognathi (5). This was folof these and related studies are reported herein. lowed by descriptions of Cu,ZnSODs from Caulobacter crescentis (6), several species of pseudomonads MATERIALS AND METHODS Bacto-tryptone and yeast extract were from Difco and bovine 1 This work was supported by grants from the Council for Tobacco Cu,ZnSOD was from Diagnostic Data Inc. Xanthine oxidase was prepared from bovine cream by the procedure of Waud et al. (27). E. coli Research-U.S.A., Inc. (2871AR2) and the U. S. Army Medical Research Office (Contract DAMD17-95-C-5065). Cu,ZnSOD was prepared as previously described (28) from strain DH5a [supE44 DlacU169 (f80 lacZ Du15) hsd R17 recA endA 2 To whom correspondence should be addressed. Fax: (919) 684-8885. gyrA96 thi1 relA1] which overproduces the Cu,ZnSOD by virtue of the sod c containing plasmid psod C2.1 (22). This strain was kindly 3 Abbreviation used: Cu,ZnSODs, copper-and zinc-containing superoxide dismutases. provided by J. A. Imlay. Cells were grown to stationary phase in 305
Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 1999
The fungal strain Humicola lutea 110 produces a mangan-and a copper zinc-containing superoxide dismutases (SOD). In this study, the purification, N-terminal sequence and spectroscopic properties of the new Cu,Zn SOD are described. The preparation of the pure metalloenzyme was achieved via treatment of the strain with acetone followed by gel and ion exchange chromatography. The protein consists of 302 amino acid residues and has a molecular mass of approximately 32 kDa, as determined by PAG electrophoresis and 3100 U mg − 1 protein-specific activity. It is a dimeric enzyme with two identical subunits of 15 950 Da, as indicated by SDS-PAGE, mass spectroscopic and amino acid analysis. The N-terminal sequence analysis of the Cu,Zn SOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Conformational stability and reversibility of unfolding of the dimeric enzyme were determined by fluorescence and circular dichroism (CD) spectroscopy. The critical temperature of deviation from linearity (T c ) of the Arrhenius plot ln (Q − 1 − 1) vs. 1/T was calculated to be 68°C and the respective activation energy for the thermal deactivation of the excited indole chromophores is 42 kcal mol − 1 . The melting temperatures (T m ) were determined by CD measurements to be 69°C for the holo-and 61°C for the apo-enzyme. The fluorescence emission of the Cu,Zn SOD is dominated by 'buried' tryptophyl chromophores. Removal of the copper-dioxygen system from the active site caused a 4-fold increase of the fluorescence quantum yield and a 10 nm shift of the emission maximum position towards higher wavelength. (P. Dolashka-Angelova) 1386-1425/99/$ -see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 -1 4 2 5 ( 9 9 ) 0 0 0 3 6 -0
Proceedings of the National Academy of Sciences, 1988
The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity. The gene was isolated from a Agtll library by using a long, unique deoxyoligonucleotide probe. The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table. The sequence of the coding and flanking regions is reported. The cloned gene was expressed and shown to be active in vivo. A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity. The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain. Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant. The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these rindings. Superoxide dismutases (SODs) are abundant enzymes present in most aerobic organisms and many anaerobic ones. Their known activity is catalysis of the disproportionation of the superoxide radical, O°, to give dioxygen and hydrogen peroxide (20 + 2H +-+02 + H202) (1, 2). They are widely assumed to provide protection in vivo against this reactive metabolic by-product, although this has not been conclusively proven (3). SOD exists in several forms in different organisms. Prokaryotes have two forms, one containing iron and one containing manganese. Eukaryotes have a manganese-containing form in the mitochondria and a copper-and zinc-containing form in the cytoplasm (Cu,Zn-SOD; EC 1.15.1.1). The Mn and Fe proteins are related to each other, whereas Cu,Zn-SOD is unrelated to either (2). The Cu,Zn-SOD protein is very well studied. The crystal structure of the bovine enzyme has been solved (4), and it and the yeast enzyme have been the subject of numerous physical and chemical studies in this and other laboratories (5). The in vivo role of Cu,Zn-SOD is less well defined. There is disagreement over whether or not O2 is a direct cause of dioxygen toxicity and, consequently, over whether SOD has an important protective function, since O2 disproportionates
Fish & Shellfish Immunology, 2010
Superoxide dismutases (SODs, EC 1.15.1.1) are one family of important antioxidant metalloenzymes involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular CuZnSOD gene of Cristaria plicata (Cp-icCuZnSOD) was identified from hemocytes by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of Cp-icCuZnSOD consisted of 891 nucleotides with a canonical polyadenylation signal sequence ATTAAA, a poly (A) tail, and an open-reading frame of 468 bp encoding 155 amino acids. The deduced amino acids of CpSOD shared high similarity with the known icCuZnSODs from other species, and several highly conserved motifs including Cu/Zn ions binding sites (His-46, His-48, His-63, His-120 for Cu 2þ binding, and His-63, His-71, His-80, Asp-83 for Zn 2þ binding), intracellular disulfide bond and two CuZnSOD family signatures were also identified in CpSOD. Furthermore, the recombinant Cp-icCuZnSOD with high enzyme activity was induced to be expressed as a soluble form by IPTG supplemented with Cu/Zn ions at 20 C for 8 h, and then was purified by using the native Ni 2þ affinity chromatography. The specific activity of the purified rCp-icCuZnSOD enzyme was 5368 U/mg, which is 2.6-fold higher than that of zebrafish Danio rerio rZSOD and 5.3-fold higher than that of bay scallop Argopecten irradians rAi-icCuZnSOD. The enzyme stability assay showed that the purified rCp-icCuZnSOD enzyme maintained more than 80% activity at temperature up to 60 C, at pH 2.0e9.0, and was resistant to 8 mol/L urea or 8% SDS. In addition, the addition of active rCp-icCuZnSOD enzmye could protect hepatocyte L02 cells from oxidative damage as assessed using an alcohol-injured human liver cell model. Crown