Artificial Modification of an Enzyme for Construction of Cross-Reactive Polyion Complexes To Fingerprint Signatures of Proteins and Mammalian Cells.

Abstract

A novel strategy for construction of cross-reactive enzyme-based sensors have been developed based on chemical modification of lysine ε-NH3(+) groups in β-galactosidase (β-Gal) from Aspergillus oryzae with various acid anhydrides. Modification of lysine side chains markedly altered the kinetic parameters of β-Gal (KM and kcat), whereas catalytic activity remained and the tertiary structure was maintained for all modified β-Gals. The addition of cationic PEGylated polymers to reactive solutions caused the formation of polyion complexes (PICs) with marked inhibition of the modified β-Gal activity. Therefore, the obtained PICs can be used to construct cross-reactive enzyme-based sensors without any purification. With addition of each analyte protein or mammalian cell to the PIC library, the modified β-Gals were partially released from PICs, and therefore the catalytic activities showed characteristic recovery fingerprints. Linear discriminant analysis (LDA) of fingerprints generated by the combination of only three PICs enabled successful discrimination of 0.5 μg/mL human plasma proteins (albumin, immunoglobulin G, transferrin, fibrinogen, α1-antitrypsin, C-reactive protein), and semiquantification of inflammatory biomarker proteins in buffer (0.3-8.1 μg/mL) and human serum (2-100 μg/mL) with comparable sensitivity for diagnosis in human blood samples. Moreover, we identified five mammalian (human, bovine, fetal bovine, horse, and rabbit) sera containing 2.5 μg/mL serum proteins, and three human cancer cell lines [A549 (lung), MG63 (bone), HuH7 (liver)] and a human mesenchymal stem cell line (UE7T-13) (1500 cells/mL) with 100% accuracy.