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  • Protein minimization: characterization of the synthetic cyclic dodecapeptide corresponding to the reactive site region of the oil rape trypsin inhibitor type-III.

Protein minimization: characterization of the synthetic cyclic dodecapeptide corresponding to the reactive site region of the oil rape trypsin inhibitor type-III.

Biochemical and biophysical research communications (2003-02-27)
Maurizio Trovato, Elena Caroli Casavola, Bruno Maras, Maria Eugenia Schininà, Paolo Costantino, Paolo Ascenzi
ABSTRACT

The design of minimal units required for enzyme inhibition is a major field of interest in structural biology and biotechnology. The successful design of the cyclic dodecapeptide corresponding to the Phe17-Val28 reactive site amino acid sequence of the low-molecular-mass trypsin inhibitor RTI-III from Brassica napus (micro-RTI-III) and of the recombinant murine dihydrofolate reductase-(DHFR-)micro-RTI-III fusion protein (DHFR-micro-RTI-III) is reported here. Micro-RTI-III was synthesized using a stepwise solid-phase approach based on the standard Fmoc chemistry, purified by RP-HPLC, and oxidatively refolded. DHFR-micro-RTI-III was expressed in Escherichia coli, purified by metal-chelate affinity chromatography, and oxidatively refolded. The affinity of micro-RTI-III for bovine trypsin (K(d)=1.6x10(-9)M) is similar to that determined for DHFR-micro-RTI-III (K(d)=6.3x10(-10)M) and native RTI-III (K(d)=2.9x10(-10)M), at pH 8.2 and 22.0 degrees C. Remarkably, micro-RTI-III protects the DHFR domain of DHFR-micro-RTI-III from trypsin digestion. Micro-RTI-III is a new minimal trypsin inhibitor and may be regarded as a tool in protein structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), Isoelectric focusing marker, pI 4.6
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), lyophilized powder
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Trypsin inhibitor from chicken egg white, Type III-O (free of ovoinhibitor)
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Trypsin inhibitor from Glycine max (soybean), solution, sterile-filtered, suitable for, suitable for cell culture
Sigma-Aldrich
Trypsin inhibitor from Phaseolus limensis (lima bean), Type II-L, crude powder
Sigma-Aldrich
Trypsin inhibitor from chicken egg white, Type II-O, Partially purified ovomucoid, containing ovoinhibitor
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), lyophilized powder
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), For use as a marker in SDS-PAGE, BioReagent
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), lyophilized powder, ~10000 U/mg
Sigma-Aldrich
Trypsin Inhibitor, Defined (1X) Solution, Animal component free, BioReagent, suitable for cell culture