Expression，purification and antimicrobial activity of SCY2 from mud crab Scylla paramamosain in yeast Pichia pastoris
PENG Hui1，2， LIU Jie1， CHEN Hui－yun1
1.College of Ocean and Earth Sciences， Xiamen University， Xiamen 361102， China； 2.State－Province Joint Engineering Laboratory of Marine Bioproducts and Technology， Xiamen 361102， China
Abstract: A 300 bp cDNA sequence of SCY2，as an anionic antimicrobial peptide recently identified from the seminal plasma of mud crab Scylla paramamosain， encoding mature peptide， was cloned into yeast Pichia pastoris by the secreted expression vector of pPIC9K，and then induced by methanol to gain more detailed information on its antimicrobial activity.The recombinant product was identified using MS－fingerprinting.The results showed that recombinant plasmid pPIC9K－SCY2 was constructed successfully， and the clone of GS115/pPIC9K－SCY2 induced by 0.5%methanol for 24 h， with a stable expressed product with molecular weight of about 11 000，whose molecular weight was consistent with the calculated molecular weight of SCY2.The purified recombinant protein was identified by mass spectrum analysis，and 6 peptide segments of recombined SCY2 were sequenced correctly using mass spectrum.The recombinant product of SCY2 was shown to have the activity against Gram－positive bacteria， without activity against Gram－negative ones(＞50 μmol/L).Synergistic antibacterial test of SCY2 with penicillin， streptomycin and tetracycline revealed that SCY2 combined with tetracycline had synergistic antibacterial function against Pseudomonas fluorescens，especially combination of SCY2 with penicillin against Pseudomonas stutzeri.The findings indicate that the combined use of low concentrations of antibiotics and antimicrobial peptides can effectively reduce the amount of antibiotics and the risk of antibiotic residues in food.
2018, Vol. 33 
