Abstract
The design of chimeric proteins is a major field of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.
Copyright 2000 Academic Press.
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Binding Sites
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Brassica / chemistry*
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Cattle
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Chromatography, Affinity
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Escherichia coli
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Kinetics
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Mice
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Molecular Sequence Data
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Plant Proteins / chemistry
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Plant Proteins / genetics
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Plant Proteins / isolation & purification
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Plant Proteins / pharmacology
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Protein Engineering
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Protein Folding
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / pharmacology*
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Tetrahydrofolate Dehydrogenase / chemistry
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Tetrahydrofolate Dehydrogenase / genetics
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Tetrahydrofolate Dehydrogenase / metabolism
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Trypsin / metabolism
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Trypsin Inhibitors / chemistry*
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Trypsin Inhibitors / genetics
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Trypsin Inhibitors / isolation & purification
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Trypsin Inhibitors / pharmacology*
Substances
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Plant Proteins
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Recombinant Fusion Proteins
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Trypsin Inhibitors
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Tetrahydrofolate Dehydrogenase
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Trypsin