Characterization of Kunitz-type inhibitor B1 performance using protein chips and AFM.

A protein chip containing 11 different proteases was used to investigate the expression of protease inhibitors induced by A. carbonarius infection of potato tubers. Kunitz-type protease inhibitors B1 (KPI-B1) recombinant proteins with trypsin/chymotrypsin protease selectivity were used as internal control and inter-assay control in protease chips targeting KPI proteins. KPI-B1 was spotted on each chip as a reference to compare fluorescence intensities between different hybridization experiments. In order to validate the use of KPI-B1 as control, we studied the performance of recombinant KPI-B1 protein. Interactions between KPI-B1 and proteases in the absence and in the presence of phenylmethylsulfonyl fluoride (PMSF) indicated that a free substrate binding pocket in protease is required for binding with the recombinant KPI-B1 inhibitor. Atomic Force Microscopy (AFM) was employed to analyse structures of protein complexes formed by KPI-B1. KPI-B1/antibody complexes have diameters of ca. 450 nm and heights of ca. 8 nm, while trypsin/KPI-B1/antibody complexes have relatively small diameters (ca. 300 nm) but very great heights (ca. 50 nm). On the basis of AFM data, trypsin-KPI-B1 complexes, instead of KPI-B1 alone, could be a better internal control for protein chip to calibrate fluorescence signals obtained from different hybridization experiments.