A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III

The design of chimeric proteins is a major held of interest in structural b iology and biotechnology. The successful design of the chimeric protein com posed by the minimized reactive site domain of the low-molecular-mass tryps in 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 b y metal-chelate affinity chromatography and oxidatively refolded. The affin ity 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 mult ifunctional and multidomain proteinase inhibitors. (C) 2000 Academic Press.