THE SPECTROSCOPIC ANALYSIS, INHIBITION AND BINDING-STUDIES DEMONSTRATE THE EQUIVALENCE OF ERYTHRINA-CAFFRA TRYPSIN-INHIBITOR AND THE RECOMBINANT SUBSTITUTION VARIANT RECSERETI

A recombinant substitution mutant (recSerETI) of the Erythrina caffi a trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein ( wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission an d circular dichroism, were indistinguishable. Furthermore, the inhibit ory activities of the two proteins regarding the inhibition of trypsin , chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (B M 06.022, t-PA deletion variant comprising the kringle 2 and the prote ase domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Fiv e repetitive cycles of guanidinium chloride (GdmCl) -induced denaturat ion-renaturation yield the native mutant protein with its inhibitory a ctivity fully restored. The only difference between the wildtype and t he mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype prote in. The pH range of stability is shifted from pH 1-9.5, for wtETI, to pH 2-9, for recSerETI; similarly the GdmCl-induced denaturation is fou nd to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; i n both cases the renaturation exhibits strong hysteresis. The mid-poin t of the thermal unfolding transition of the mutant protein is at simi lar to 65 degrees C, as compared to similar to 75 degrees C for the wi ldtype protein. (C) 1998 Elsevier Science B.V. All rights reserved.