Expression of Swimming Crab C-type Lysozyme in Pichia pastoris and Its Bacteriostatic Activity

doi:10.13560/j.cnki.biotech.bull.1985.2018-0241

Abstract

Abstract:

C-type lysozyme is an innate immune protein existing in different tissues of all organisms,thus it is a favorable candidate for developing natural bacteriostatic agent. In this study,mature peptide of C-type lysozyme from crustacean swimming crab（Portunus trituberculatus）was the object of recombinant DNA expression. Firstly,cDNA fragment encoding the C-type lysozyme mature peptide was cloned by RT-PCR,and was named as “ptlyC”. The ptlyC was ligated to pPICZαA vector to construct recombinant expression vector pPICZαA-ptlyC;then the pPICZαA-ptlyC was transformed into competent Pichia pastoris X-33 cells. Yeast transformants containing multi-copy gene insertion were screened by high concentration of zeocin. Recombinant ptlyC was induced with 1.0%（V/V）methanol at 28℃,250 r/min and pH6.0 for 72 h. The culture medium supernatant containing recombinant ptlyC was subjected to immobilized metal affinity chromatography（IMAC）for purification,and the molecular mass of the purified ptlyC was about 25.3 kD. Micro liquid-bacterial colony notation demonstrated that the culture medium supernatant containing recombinant ptlyC had a bacteriostatic effect on gram-positive Bacillus subtilis. Further,agar well diffusion assays confirmed that the purified recombinant ptlyC inhibited gram-negative Pseudomonas aeruginosa,Vibrio parahaemolyticus and Salmonella lignieres.

Key words: Protunus trituberculatus, C-type lysozyme, Pichia pastoris, recombinant DNA expression, bacteriostatic activity

WANG Qiang-hou, TAO Yan, CUI Xu, YAN Qian-qian, ZHANG Ya-li. Expression of Swimming Crab C-type Lysozyme in Pichia pastoris and Its Bacteriostatic Activity[J]. Biotechnology Bulletin, 2018, 34(10): 135-142.

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