Biosynthesis of Crucian Carp (Carassius auratus) c-type Lysozyme Based on Recombinant Pichia pastoris and Its Characteristics
SONG Yu-Fang1, TAO Yan1,2,*, XIE Jing1,2, QIAN Yun-Fang1,2
1 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China; 2 Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China;
Abstract：As key immune protein in innate immune mechanism of crucian carp (Carassius auratus), c-type lysozyme is a good candidate for the development of green feed additives or natural antimicrobial agents. In present study, the cDNA encoding crucian carp c-type lysozyme was cloned by reverse transcription PCR (RT-PCR). Subsequently, 3 times of PCR were performed to add the XhoⅠrestriction endonuclease site, Kex2 signal peptidase cleavage site and a 6×His tag to its 5' end, and the XbaⅠrestriction endonuclease site and stop codon to its 3' end; the obtained target gene was named CrCLm (crucian carp c-type lysozyme mature peptide). Using pPICZɑA as expression vector and Pichia pastoris X-33 as engineering strain, the recombinant P. pastoris strain X-33/pPICZɑA-CrCLm was constructed. After the recombinant strain was induced at initial pH 6.0, 28 ℃ and 250 r/min for 96 h with 1.0% methanol, the obtained recombinant protein was purified by a nickel ion affinity chromatography column. The purified protein was used for Western blot and LC-MS/MS analysis. On the other hand, the antibacterial activity and stability for the recombinant protein were determined. The results indicated that under above fermentation conditions, the recombinant protein with a yield of 18.3 mg/L was obtained, and it was identified by LC-MS/MS analysis as expected recombinant CrCLm with a molecular weight of 14.5 kD. The results of antibacterial test showed that the recombinant CrCLm had obvious activity against Gram-positive Staphylococcus aureus, Bacillus subtilis, B. cereus and Listeria monocytogenes, and Gram-negative Escherichia coli, Salmonella, Vibrio parahaemolyticus and Pseudomonas aeruginosa. In addition, different acidic environments, high temperature treatment and hydrolysis of proteinase (such as trypsin, pepsin and proteinase K) did not affect its antibacterial activity. The recombinant P. pastoris strain constructed in the present study can effectively synthesize CrCLm with obvious antibacterial activity and good stability, which could provide technical support for its large-scale preparation and application.
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