Recombinant DNA Expression of I-Type Lysozyme of Clam Cyclina sinensis in Yeast Pichia pastoris
FENG Fu′ai1, ZHAO Zhen1, TAO Yan1,2, XIE Jing1,2, QIAN Yunfang1,2
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：I-type lysozyme as a cysteine-rich protein immune factor mainly expressed in invertebrate has strong activity against pathogenic microorganisms. In the present study, the cDNA encoding I-type lysozyme of clam Cyclina sinensis was cloned by RT-PCR, known as CslyI gene. Using pPICZαA as expression vector and yeast Pichia pastoris X-33 as engineering bacteria to construct recombinant strain X-33/pPICZαA-CslyI. A high-copy recombinant strain was obtained by using the culture medium containing high concentrations of zeocin. The strain created protein-like expression products after 72 h induced by 1% methanol at 28 ℃, and 250 r/min. The expressed product was identified by Western-blot and MALDI-TOF/TOF mass spectrometry, indicating that it was the expected recombinant CslyI. The bacteriostatic tests demonstrated that the recombinant CslyI had obvious activities against pachogens Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus. In addition, it also showed a good thermal stability. The findings provide a biosynthetic technology approach based on recombinant yeast P. pastoris for the preparation of I-type lysozyme derived from invertebrate.
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2022, Vol. 41 
