Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris.
Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China.
Tingting
T
Wang
Yongping
Y
Xu
Wenjia
W
Liu
Yongxin
Y
Sun
Liji
L
Jin
D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D008837:Micrococcus / Q000187:drug effects; D009113:Muramidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D047528:Stichopus / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D009113:Muramidase; D000432:Methanol