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| Recombinant Expression of Common Carp (Cyprinus carpio) c-type Lysozyme in Pichia pastoris and Its Antibacterial Activity |
| YAN Qian-Qian, TAO Yan*, LI Wen, XIE Jing, QIAN Yun-Fang |
| College of Food Science and Technology/Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai Ocean University, Shanghai 201306, China |
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Abstract The c-type lysozyme is a key immune protein to resist bacterial infection for the fish with innate immune system. Common carp (Cyprinus carpio) c-type lysozyme has some characteristics that are common in fish c-type lysozyme: Its N-terminal is a signal peptide sequence, and its C-terminal region is a mature peptide sequence. Glu35 and Asp51 are 2 amino acid residues located in the mature peptide and form the active site of the c-type lysozyme. In addition, 8 conserved cysteine residues exist in the mature peptide and form 4 pairs of disulfide bonds in spatial structure which are related to the activity and stability of the c-type lysozyme. In the present study, the cDNA encoding common carp c-type lysozyme mature peptide was cloned by reverse transcription PCR (RT-PCR), and the 381 bp cDNA encoded the mature peptide consisting of 127 residues. PCR were performed to add XhoⅠ restriction site and 6×His-tag to its 5' end, and XbaⅠ site to its 3' end. This target gene was named CLYc (common carp c-type lysozyme), and ligated to pPICZαA vector and electronically transformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using high-concentration zeocin. The target protein was induced for 120 h with 1.5% methanol at 29 ℃, 250 r/min. The recombinant protein with molecular weight of 15.4 kD was purified by immobilized metal ion affinity chromatography (IMAC); Western blot and MALDI-TOF/TOF mass spectrometry analysis proved that this recombinant protein was the expected recombinant CLYc, and its expression yield was about 40 mg/L. The antimicrobial activity of the culture medium supernatant containing recombinant CLYc was tested on Gram-positive (including Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (including Escherichia coli and Vibrio parahemolyticus) bacteria using micro liquid-bacterial colony notation, demonstrating that the crude culture medium supernatant had notable antimicrobial activity against these bacteria, implying the potential use of cell-free P. pastoris medium in fish feed without the need for further peptide purification. Furthermore, the minimum inhibitory concentration (MIC) of purified recombinant CLYc was determined on these bacteria and Bacillus subtilis, indicating that the recombinant CLYc had the same MIC for L. monocytogenes, B. subtilis, E. coli and V. parahemolyticus. The results of this study provide a technical approach for the development and production of natural antibacterial agents from fish.
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Received: 24 February 2019
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Corresponding Authors:
ytao@shou.edu.cn
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