Enhanced Expression and Functional Characterization of the Recombinant Putative Lysozyme-PMAP36 Fusion Protein
Zhili Rao1,8, So Young Kim1,8, Md Rashedunnabi Akanda2,3, Su Jin Lee4, In Duk Jung4, Byung-Yong Park2, Seralathan Kamala-Kannan1,5, Jin Hur6, and Jung Hee Park1,7,*
1Division of Biotechnology, College of Environmental & Bioresources Sciences, 2College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University, Iksan 54596, Korea, 3Department of Pharmacology and Toxicology, Sylhet Agricultural University, Sylhet 3100, Bangladesh, 4Department of Immunology, Laboratory of Dendritic Cell Differentiation and Regulation, School of Medicine, Konkuk University, Chungju 27478, Korea, 5Advanced Institute of Environment and Bioscience, College of Environmental & Bioresources Sciences, 6Veterinary Public Health, College of Veterinary Medicine, 7Safety, Environment and Life Science Institute, College of Environmental and Bioresources Sciences, Chonbuk National University, Iksan 54596, Korea, 8These authors contributed equally to this work.
*Correspondence: junghee.park@jbnu.ac.kr
Received September 1, 2018; Revised December 24, 2018; Accepted January 27, 2019.; Published online March 28, 2019.
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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.
Keywords: circular dichroism spectroscopy, hemolytic activity, minimum inhibitory concentration, procine myeloid antimicrobial peptiede, trasmission electron microscope

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