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

Role of TLR5 and Flagella in <i>Bacillus</i> Intraocular Infection

<div><p><i>B. cereus</i> possesses flagella which allow the organism to migrate within the eye during a blinding form of intraocular infection called endophthalmitis. Because flagella is a ligand for Toll-like receptor 5 (TLR5), we hypothesized that TLR5 contributed to endophthalmitis pathogenesis. Endophthalmitis was induced in C57BL/6J and TLR5−/− mice by injecting 100 CFU of <i>B. cereus</i> into the mid-vitreous. Eyes were analyzed for intraocular bacterial growth, retinal function, and inflammation by published methods. Purified <i>B. cereus</i> flagellin was also injected into the mid-vitreous of wild type C57BL/6J mice and inflammation was analyzed. TLR5 activation by <i>B. cereus</i> flagellin was also analyzed <i>in vitro. B. cereus</i> grew rapidly and at similar rates in infected eyes of C57BL/6J and TLR5−/− mice. A significant loss in retinal function in both groups of mice was observed at 8 and 12 hours postinfection. Retinal architecture disruption and acute inflammation (neutrophil infiltration and proinflammatory cytokine concentrations) increased and were significant at 8 and 12 hours postinfection. Acute inflammation was comparable in TLR5−/− and C57BL/6J mice. Physiological concentrations of purified <i>B. cereus</i> flagellin caused significant inflammation in C57BL/6J mouse eyes, but not to the extent of that observed during active infection. Purified <i>B. cereus</i> flagellin was a weak agonist for TLR5 <i>in vitro</i>. These results demonstrated that the absence of TLR5 did not have a significant effect on the evolution of <i>B. cereus</i> endophthalmitis. This disparity may be due to sequence differences in important TLR5 binding domains in <i>B. cereus</i> flagellin or the lack of flagellin monomers in the eye to activate TLR5 during infection. Taken together, these results suggest a limited role for flagellin/TLR5 interactions in <i>B. cereus</i> endophthalmitis. Based on this and previous data, the importance of flagella in this disease lies in its contribution to the motility of the organism within the eye during infection.</p></div

Whole eye histology of experimental <i>B. cereus</i> endophthalmitis.

<p>C57BL/6J and TLR5−/− mouse eyes were injected with 100 CFU <i>B. cereus</i>. Whole globes were harvested and processed for hematoxylin and eosin staining. Infected eyes of both groups had significant inflammation by 12 h postinfection, suggesting that the absence of TLR5 did not greatly impact intraocular inflammation. Sections are representative of 4 eyes per group. Magnification, 10X.</p

Multiple sequence alignments of <i>S. typhimurium</i>, <i>B. cereus</i>, <i>B. thuringiensis</i>, and <i>B. anthracis</i> flagellins.

<p>The amino acid sequences of <i>B. cereus</i> (Bc) and <i>S. typhimurium</i> (St) (top) and <i>Bc, B. thuringiensis</i> (Bt), and <i>B. anthracis</i> (Ba) (bottom) were aligned by ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100543#pone.0100543-AndersenNissen2" target="_blank">[71]</a>, focusing on regions important for IL8 activity (Region 1) and TLR5 stimulation and recognition (Regions 2 and 3). Asterisks and red letters identify amino acids conserved between Bc and St sequences or among Bc/Ba/Bt sequences. Blue amino acids have strongly similar properties, while green amino acids have weakly similar properties. Bc and St had 81% conserved residues in Region 1, 62.5% conserved residues in Region 2, and 25% conserved residues in Region 3. Bc, Ba, and Bt had 86% conserved residues in Region 1, 62.5% conserved residues in Region 2, and 44% conserved residues in Region 3.</p

Influence of TLR5 on bacterial growth during experimental <i>B. cereus</i> endophthalmitis.

<p>C57BL/6J wild type and TLR5−/− mouse eyes were injected with 100 CFU <i>B. cereus</i>. Eyes were harvested, homogenized, and analyzed for bacterial growth. Overall, <i>B. cereus</i> grew to similar concentrations in infected eyes of TLR5−/− and C57BL/6J mice, suggesting that the absence of TLR5 did not influence the overall growth of <i>B. cereus</i> in the eye. Values represent the mean log₁₀ CFU±SD of N≥4 eyes per time point for at least 2 separate experiments. *P≤0.05.</p

Influence of TLR5 on infiltration of PMN into mouse eyes during experimental <i>B. cereus</i> endophthalmitis.

<p>C57BL/6J and TLR5−/− mouse eyes were injected with 100 CFU <i>B. cereus</i>. PMN infiltration was estimated by quantifying MPO in whole eyes by sandwich ELISA. MPO levels were similar in these groups at all times points postinfection (P≥0.17), suggesting that the absence of TLR5 did not alter the PMN response during infection. Values represent the mean ±SD for N≥4 per group for at least 2 separate experiments. *P≤0.05.</p

Influence of TLR5 on retinal function during experimental <i>B. cereus</i> endophthalmitis.

<p>C57BL/6J wild type and TLR5−/− mouse eyes were injected with 100 CFU <i>B. cereus</i>. Retinal function was assessed by electroretinography (ERG). <b>A</b>) A-wave amplitudes were slightly greater in C57BL/6J infected eyes at 12 h postinfection (P = 0.02), while B-wave amplitudes were greater in TLR5−/− infected eyes at 8 h postinfection (P = 0.008). By 12 hours postinfection, A-wave and B-wave amplitudes retained in both groups decreased to approximately 20% or less in infected eyes, indicating significant retinal function loss in both groups of mice regardless of the presence of TLR5. Values represent the mean ±SD of N≥4 eyes per time point for at least 2 separate experiments. *P≤0.05. <b>B</b>) Representative averaged waveforms from wild type (WT) and TLR5−/− mice at 12 h postinfection, with one eye infected and the contralateral eye serving as the uninfected control. Representative of N≥4 eyes per time point.</p

Influence of TLR5 on proinflammatory mediator expression during experimental <i>B. cereus</i> endophthalmitis.

<p>C57BL/6J and TLR5−/− mouse eyes were injected with 100 CFU <i>B. cereus</i>. Ocular proinflammatory cytokines and chemokines were analyzed by sandwich ELISA. Overall, similar levels of TNFα, KC, IL6, and IL1β were synthesized in infected eyes of C57BL/6J mice compared with that in infected eyes of TLR5−/− mice, suggesting that the absence of TLR5 did not alter the inflammatory mediator response during infection. Values represent the mean ±SD for N≥4 per group for at least 2 separate experiments. *P≤0.05.</p

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