Expression and purification of active recombinant cathepsin C (dipeptidyl aminopeptidase I) of kuruma prawn Marsupenaeus japonicus in insect cells.
Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37 degrees C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-beta-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.
Qiu
Gao-Feng
GF
E-Institute of Shanghai Universities (EISU) Aquaculture Division and Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology, College of Life Science, Shanghai Ocean University, Shanghai, China. gfqiu@shou.edu.cn
Feng
Hai-Yang
HY
Yamano
Keisuke
K
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D020789:Cathepsin C / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007915:Lepidoptera / Q000821:virology; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D020789:Cathepsin C
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