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

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 =1.6×10 −9 M ) is similar to that determined for DHFR-micro-RTI-III ( K d =6.3×10 −10 M ) and native RTI-III ( K d =2.9×10 −10 M ), at pH 8.2 and 22.0 °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.