ABSTRACT
Immunosensors based on gold electrodes (electrochemical) or gold discs (optical) modified with 1,6-hexanedithiol, gold nanorods and Anti-His (C-term) monoclonal antibody F(ab') fragment are described. The antigen detected by the sensing platform is a recombinant histidine-tagged silk proteinase inhibitor (rSPI2-His(6)). Electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques were used as methods for detection of the antigen. This approach allows to detect the antigen protein in concentration of 10 pg per mL (0.13 pM) of culture medium. The immunosensor shows good reproducibility due to covalent immobilization of F(ab') fragments to gold nanorods layer.
Subject(s)
Biosensing Techniques/methods , Culture Media/chemistry , Gold/chemistry , Histidine/immunology , Immunoglobulin Fab Fragments/chemistry , Insect Proteins/analysis , Biosensing Techniques/instrumentation , Dielectric Spectroscopy/methods , Electrodes , Gold/pharmacology , Histidine/chemistry , Histidine/genetics , Immunoassay , Immunoglobulin Fab Fragments/pharmacology , Insect Proteins/chemistry , Insect Proteins/genetics , Nanotubes/chemistry , Pichia , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Surface Plasmon Resonance/methodsABSTRACT
Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.
