Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

S-Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Impact of Different Metabolic Uncouplers on the Specific Degradation Rate of Toluene in a Differential Biofiltration Reactor

In this work, a differential biofiltration reactor was used to explore the potential of metabolic uncouplers to improve pollutant (toluene) degradation rates. Metabolic uncouplers were reported to reduce the cell mass in activated sludge systems, but are untested in biofilters and the current work is the first to report the impact of different metabolic uncouplers in a biofilter. Initially soil was used as a biofilter bed and later experiments were conducted in pure cultures in a biofilm reactor. A simple diffusion system was developed to generate the desired concentration of toluene to the system. Gas chromatography and a carbon dioxide analyzer were connected online to the reactor which improved the precision of the data collected and also the robustness of the measurements. Preliminary experiments including effect of substrate concentration, different nutrients and temperature were done to optimize the conditions before starting the metabolic uncoupler screening studies in soil. Based on the results, inlet toluene concentration between 180 ppm and 250 ppm was used throughout the studies. Also it was found that the toluene degraders were nitrogen limited. Temperature studies showed that the elimination capacity (EC) increased with increasing temperature, from 34 ± 1.4 g.m-3.h-1 to 49.8 ± 2.6 g.m-3.h-1 for temperatures of 20 to 45 oC, respectively. Nine potential metabolic uncouplers were screened in batch serum bottles. The nine uncouplers tested were dinitrophenol (dNP), p-nitrophenol (pNP), benzoic acid (BA), carbonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP), carbonylcyanide m-chloromethoxy phenylhydrazone (CCCP), pentachlorophenol (PCP), malonic acid (MA), m-chlorophenol (mCP) and 2, 4, 6-trichlorophenol (TCP). Other than dNP and pNP (nitrogen containing uncouplers), seven other uncouplers were further tested in the differential biofilter reactor. Only PCP and TCP increased the toluene degradation rate significantly. PCP increased the toluene degradation rate by 35% at 140 µM, whereas 4051 µM TCP increased the rate by 18%. Though FCCP behaved as a classical uncoupler when compared with others, the EC increase was not significant. Five toluene degraders were isolated from soil subjected to toluene and were identified using 16s rDNA/18s rDNA analysis. Out of five, two potential toluene degraders, Stenotrophomonas maltophilia and Pseudomonas putida were used to develop a biofilm reactor. PCP, TCP and CCCP were tested in the biofilm reactors and found that PCP increased the surface elimination capacity (SEC) by 85% at 140 µM in S. maltophilia biofilm reactor and CCCP increased the SEC by 27% at 1 µM in P. putida biofilm reactor. Finally a simple model was developed to calculate the energy uncoupling coefficient for non-growth systems like ours to quantitatively represent the uncoupling mechanism

Site directed mutagenesis of human Interleukin-2 gene to increase the stability of the gene product : A Bioinformatics Approach

Interleukin-2 (IL-2) is an immunoregulatory cytokine whose biological effects are mediated through interaction with specific receptors on the surface of target cells. Due to its presumed role in generating a normal immune response, IL-2 is being evaluated for the treatment of a variety of tumors, in addition to infectious diseases. Main drawback of human IL-2 is that the molecule is relatively unstable. Therefore, with the objective of increasing the stability of the molecule, site directed mutagenesis of human IL-2 gene was carried out. Early studies indicated that mutations at three Cysteine residues (58, 105, 125) which are in the active sites of human IL-2 resulted in the reduced stability as well as the biological activity of the molecule. Therefore, mutations were carried out at the positions of amino acid other than the receptor binding sites at 111Valine to Arginine, 117Lysine to Glutamine and 133 Threonine to Asparagine of the human sequence by comparing it with the bovine sequence which has higher stability than the human counterpart, using SWISS PDB tool. To understand the biological activity of the mutated IL-2, energy minimization studies were carried out using SWISS-PDB. Docking studies were performed to check the reliability of the results using HEX DOCK, ARGUS LAB and PATCH DOCK between the IL-2 receptor and its mutated Ligand. These docking results also confirmed that the reliability of these mutated IL-2 gene. Stability, half life and ADME characteristics of these mutants can be studied in a detailed manner in the in vivo studies.

Site directed mutagenesis of human Interleukin-2 gene to increase the stability of the gene product : A Bioinformatics Approach

Interleukin-2 (IL-2) is an immunoregulatory cytokine whose biological effects are mediated through interaction with specific receptors on the surface of target cells. Due to its presumed role in generating a normal immune response, IL-2 is being evaluated for the treatment of a variety of tumors, in addition to infectious diseases. Main drawback of human IL-2 is that the molecule is relatively unstable. Therefore, with the objective of increasing the stability of the molecule, site directed mutagenesis of human IL-2 gene was carried out. Early studies indicated that mutations at three Cysteine residues (58, 105, 125) which are in the active sites of human IL-2 resulted in the reduced stability as well as the biological activity of the molecule. Therefore, mutations were carried out at the positions of amino acid other than the receptor binding sites at 111Valine to Arginine, 117Lysine to Glutamine and 133 Threonine to Asparagine of the human sequence by comparing it with the bovine sequence which has higher stability than the human counterpart, using SWISS PDB tool. To understand the biological activity of the mutated IL-2, energy minimization studies were carried out using SWISS-PDB. Docking studies were performed to check the reliability of the results using HEX DOCK, ARGUS LAB and PATCH DOCK between the IL-2 receptor and its mutated Ligand. These docking results also confirmed that the reliability of these mutated IL-2 gene. Stability, half life and ADME characteristics of these mutants can be studied in a detailed manner in the in vivo studies.

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which Detchanamurthy, S. et al. 7 modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

S-Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Suppression of Arabidopsis mediator subunit-encoding MED18 confers broad resistance against DNA and RNA viruses while MED25 is required for virus defense

Mediator subunits play key roles in numerous physiological pathways and developmental processes in plants. Arabidopsis Mediator subunits, MED18 and MED25, have previously been shown to modulate disease resistance against fungal and bacterial pathogens through their role in jasmonic acid (JA) signaling. In this study, Arabidopsis mutant plants of the two Mediator subunits, med18 and med25, were tested against three ssRNA viruses and one dsDNA virus belonging to four different families: Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV), Alternanthera mosaic virus (AltMV), and Cucumber mosaic virus (CMV). Although both subunits are utilized in JA signaling, they occupy different positions (Head and Tail domain, respectively) in the Mediator complex and their absence affected virus infection differently. Arabidopsis med18 plants displayed increased resistance to RNA viral infection and a trend against the DNA virus, while med25 mutants displayed increased susceptibility to all viruses tested at 2 and 14 days post inoculations. Defense marker gene expression profiling of mock- and virus-inoculated plants showed that med18 and med25 mutants exhibited an upregulated SA pathway upon virus infection at 2 dpi for all viruses tested. JA signaling was also suppressed in med18 plants after virus infection, independent of which virus infected the plants. The upregulation of SA signaling and suppression of JA signaling in med18 may have led to more targeted oxidative burst and programmed cell death to control viruses. However, the susceptibility exhibited by med25 mutants suggests that other factors, such as a weakened RNAi pathway, might play a role in the observed susceptibility. We conclude that MED18 and MED25 have clear and opposite effects on accumulation of plant viruses. MED18 is required for normal virus infection, while MED25 is important for defense against virus infection. Results from this study provide a better understanding of the role of Mediator subunits during plant-virus interactions, viral disease progression and strategies to develop virus resistant plants