Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (K-a = 8.9 x 10(5) M-1) is about 600-fold lower than that for bovine trypsin (K-a = 5.1 x 10(8) M-1); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (K-a = 7.1 x 10(8) M-1). Amino acid sequence alignment of BLCT, human beta II-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences

Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography

Protein expression in transgenic plants is considered one of the most promising approaches for producing pharmaceutical proteins. As has happened with other recombinant protein production schemes, the downstream processing (dsp) of these proteins produced in plants is key to the technical and economic success of large-scale applications. Since dsp of proteins produced transgenically in plants has not been extensively studied, it is necessary to broaden the investigation in this field in order to more precisely evaluate the commercial feasibility of this route of expression. In this work, we studied the substitution of an IMAC chromatographic step, described in previous work (Azzoni et al., 2002), with ion-exchange chromatography on SP Sepharose Fast Flow resin as the second step in the purification of recombinant aprotinin from transgenic maize seed. The main goal of this second purification step is to separate the recombinant aprotinin from the native corn trypsin inhibitor. Analysis of the adsorption isotherms determined at 25°C under different conditions allowed selection of 0.020 M Tris pH 8.5 as the adsorption buffer. The cation-exchange chromatographic process produced a high-purity aprotinin that was more than ten times more concentrated than that generated using an IMAC step