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Insight into Structural Aspects of Histidine 284 of Daphnia magna Arginine Kinase
Rao, Zhili,Kim, So Young,Li, Xiaotong,Kim, Da Som,Kim, Yong Ju,Park, Jung Hee Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.9
Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK.
Rao, Zhili,Kim, So Young,Akanda, Md Rashedunnabi,Lee, Su Jin,Jung, In Duk,Park, Byung-Yong,Kamala-Kannan, Seralathan,Hur, Jin,Park, Jung Hee Korean Society for Molecular and Cellular Biology 2019 Molecules and cells Vol.42 No.3
The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.
Zhili Rao,김소영,Md Rashedunnabi Akanda,이수진,정인덕,박병용,카말라칸,허진,박정희 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.3
The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration (128μM) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.
( Ja Young Moon ),( Won Kyong Kim ),( Zhili Rao ),( So Young Kim ),( Jung Hee Park ),( Jin Hur ) 대한인수공통전염병학회 2016 창립총회 및 학술대회 초록집 Vol.2016 No.1
Introduction: Salmonella entericas erovar Typhimurium (S. Typhimurium) mainly cause gastroenteritis in domestic animals as well as humans. S. Typhimurium in mouse infection models can cause enteric fever with similar symptoms observed in humans after S. Typhi infections. The objective of this study is summarized as first to express and purify recombinant PMAP36-lysozyme fusion protein using overexpression system. Salmonella Typhimurium isolate from Korean chicken which is used along with expressed and purified recombinant PMAP36-lysozyme fusion protein to construct Salmonella Typhimurium ghost vaccine candidate. Then constructed Salmonella Typhimurium ghost vaccine candidate used to investigate the protection efficacy in the mouse model. Methods: In order to generation of Salmonella Typhimurium ghost vaccine candidate, the recombinant PMAP36-lysozyme fusion protein expression system was constructed using Escherichia coli overexpression system which results into fusion protein expression. The expressed fusion protein was confirmed by Western blot analysis. S. Typhimurium was lysed by the fusion protein and the lysed cells were confirmed via transmission electron microscope. The S. Typhimurium lysate was evaluated as a vaccine candidate. Forty BALB/c mice were equally divided into 4 groups. Group A were orally inoculated with 100 μl of sterile PBS. And groups B-D mice were orally immunized with approximately 1.0 × 10<sup>7</sup>cells, 1.0 × 10<sup>8</sup> cells and 1.0 × 10<sup>9</sup> cells of the Salmonella vaccine candidate in 20 μl, respectively. Results: Salmonella Typhimurium-outer membrane proteins (OMPs)-specific serum IgG titers were considerably higher in groups B-D than in group A. The levels of TNF-α and IFN-γ in groups B-D than in group A were significantly higher. Following challenge with wild-type Salmonella Typhimurium isolate, all immunized groups showed the significant level of protection compared to group A. Especially, the highest protection against the challenge strain were shown in group D. Conclusion: Overall, these results shows that the vaccine candidate by GI24 can be used as an efficient vaccine for protection of systemic infections of virulent Salmonella Typhimurium.