Expression of a cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea).
|Fakulta / Pracoviště MU|
|Popis||Our research is focused primarily on expression and characterization of cystatin produced by platyhelminth Eudiplozoon nipponicum. This blood-feeding ectoparasite of Cyprinus carpio (common carp) is a representative of the family Diplozooidae (Monogenea). Cystatins – cysteine peptidase inhibitors are produced by a wide range of organisms and belong to bioactive molecules with immunomodulatory and inhibitory properties. The transcriptomic data of E. nipponicum were analyzed for the presence of cystatin sequences. The partial nucleotide sequence was identified and the whole gene sequence was obtained by PCR, 3’/5’ RACE PCR and sequencing using cDNA as a template. Gene coding 98 amino acid cystatin of E. nipponicum with predicted molecular weight 10.85 kDa and theoretical pI 6.27 was inserted into pET19b expression vector and the obtained construct was transferred into E. coli competent cells (BL 21 strain). Expression of recombinant cystatin molecule was induced by adding IPTG to the cultivation media. The success of production was confirmed by mass spectrometry in a dominant protein band after the fractionation of the E. coli derived protein mixture by SDS-PAGE. Recombinant protein was produced in the insoluble form incorporated into inclusion bodies. The cystatin from E. nipponicum will be subsequently solubilized and tested for its functional and structural properties.|