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Title
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Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.
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Authors
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E.A.Auerswald,
R.Morenweiser,
C.P.Sommerhoff,
G.P.Piechottka,
C.Eckerskorn,
L.G.Gürtler,
H.Fritz.
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Ref.
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Biol Chem Hoppe Seyler, 1994,
375,
695-703.
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PubMed id
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Abstract
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A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C),
was designed, cloned and expressed. The gene assembled via 6 oligonucleotides
contains linker sequences, stop codons and internal restriction recognition
sites for cloning, expression and cassette mutagenesis. Periplasmatic expression
products could not be detected in Escherichia coli (E. coli), but strong
expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10
mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The
secreted material was isolated after cross-flow filtration and purified by
cation exchange chromatography. The recombinant material proved to be pure and
homogeneous by electrophoretic and chromatographic analyses. Amino acid
sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray
ionization mass spectrometry confirmed that rLDTI-C was processed correctly and
that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism)
spectrum of the recombinant inhibitor is typical for a small folded protein.
rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and
human mast cell tryptase display equilibrium dissociation constants which are
nearly identical to those with the natural inhibitor. Remarkably, the inhibitor
blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.
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