Graduate Thesis Or Dissertation
 

Rainbow trout cystatin C : gene expression, heterologous production and characterization

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ms35tc21c

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  • Rainbow trout cystatin C cDNA has been isolated from trout liver. The full-length cystatin cDNA (674 bp) included the 5'untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA defines 132 amino acid residues. Comparison of the amino acid sequence with those of family 2 cystatins indicates that the 21 amino acids at the N-terminal end is a signal peptide necessary for cystatin secretion, and the remaining 111 amino acids represent mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds producing a molecule with the properties of a family 2 cystatin. Trout cystatin C gene expression was analyzed by Northern blot. This gene is expressed at various levels in all tissues examined. This difference may reflect differences in degree of regulation of cysteine proteinase activities. A high level of trout cystatin C expressed in trout hepatic tissue or cell cultures suggested that cystatin C expression might be related to tumorigenesis. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. Trout cystatin C was expressed in E. coli at a yield of 3-5 mg/L culture, but no inhibitory activity was detected for the untreated recombinant protein. However, after refolding, recombinant cystatin C displayed inhibitory activity against papain. The dissociation constant of recombinant cystatin C against papain is 1.2 x 10⁻⁶ nM, similar to that of human cystatin C. Trout cystatin C was also expressed in yeast cells, but no inhibitory activity was detected either. No cystatin C was secreted in the yeast expression system using either the trout cystatin C secretion signal, or the yeast invertase secretion signal. The expression levels of trout cystatin C in our expression systems are still low for industrial requirements. Therefore, further investigation will be needed to construct more efficient expression systems and vectors for trout cystatin C heterologous production.
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