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 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.
Trovato
Maurizio
M
Dipartimento di Genetica e Biologia Molecolare Charles Darwin, Università di Roma La Sapienza, Italy.
Casavola
Elena Caroli
EC
Maras
Bruno
B
Schininà
Maria Eugenia
ME
Costantino
Paolo
P
Ascenzi
Paolo
P
D001665:Binding Sites; D029688:Brassica napus / Q000737:chemistry*; D015195:Drug Design; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology*
D010456:Peptides, Cyclic; D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; C499355:trypsin inhibitor type II-S protein, Glycine max; C069599:trypsin inhibitor protein, Brassica napus