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

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 lowmolecular-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. DHFRmicro- RTI-III was expressed in Escherichia coli, purified bymetal-chelate affinity chromatography , and oxidatively refolded. The affinityof micro-RTI-III for bovine trypsin (Kd ¼ 1:6 109 M) is similar to that determined for DHFR-micro-RTI-III (Kd ¼ 6:3 1010 M) and native RTI-III (Kd ¼ 2:9 1010 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.