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������ 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. / Graduation date: 1999
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/33640 |
| Date | 17 July 1998 |
| Creators | Li, Fugen |
| Contributors | Barnes, David W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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