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Title
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Purification, characterization and biological evaluation of recombinant leech-derived tryptase inhibitor (rLDTI) expressed at high level in the yeast Saccharomyces cerevisiae.
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Authors
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G.Pohlig,
G.Fendrich,
R.Knecht,
B.Eder,
G.Piechottka,
C.P.Sommerhoff,
J.Heim.
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Ref.
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Eur J Biochem, 1996,
241,
619-626.
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PubMed id
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Abstract
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An efficient expression/purification procedure has been developed which allows
the production of pure, biologically active recombinant leech-derived tryptase
inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene
for LDTI was generated synthetically from three overlapping oligonucleotides by
PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under
the control of the copper-inducible CUP1 promoter and fused to the invertase
signal sequence (SUC2). The entire expression cassette was inserted into the
yeast high-copy vector pDP34. Appropriate host strains transformed with the
expression plasmid secreted rLDTI into the medium upon copper addition.
Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor
with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared
to be modified by glycosylation and the unglycosylated material showed
heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI
species lacking either the terminal Asn46 or in addition the penultimate Leu45
were isolated. The C-terminally truncated variants were eliminated using a S.
cerevisiae host strain disrupted in the structural genes of carboxypeptidases
yscY and ysca, thus identifying these proteases as being responsible for the
degradation of rLDTI. Mature rLDTI was purified in high yields from the culture
supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange
chromatography and reverse-phase HPLC. The recombinant protein is at least 98%
pure, based on HPLC and capillary electrophoresis, and is fully biologically
active. Structural identity with the authentic leech protein was confirmed by
sequence analysis and molecular-mass determination. The purified protein was
tested for its ability to inhibit tryptase and trypsin in vitro and to interfere
with the tryptase-induced proliferation of human fibroblasts and keratinocytes.
Recombinant LDTI appears to be as potent as the authentic leech protein,
exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against
human tryptase and bovine trypsin, respectively. The tryptase-induced
proliferation of human fibroblasts and keratinocytes was inhibited with
half-maximum values of approximately 0.1 nM and approximately 1 nM,
respectively. The availability of the recombinant material will allow evaluation
of the concept of tryptase inhibition in various disease models and to test the
therapeutic potential of LDTI in mast-cell-related disorders.
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