Tailoring molecular sensing for peptide displaying engineered enzymes

Abstract

Regulable enzymes displaying foreign peptides are valuable instruments for molecular targeting and fast analyte detection in homogeneous assays. Both the specificity and the intensity of the signal generated by the sensor are critical parameters that can be manipulated by trial-and-error protein engineering in the vicinity of the active site. An alternative approach is presented to enhance signal-background ratio in β-galactosidase-based molecular sensors by an optimisation of the sensing conditions. The screening of the enzymatic response in a set of engineered enzymes has revealed an antibody-dependent increase in their specific activity up to 500% for the enzyme, HD72CA, that is reached with 0.25 pmol enzyme per reaction in presence of 1.75 mM substrate. This value, much higher than 200% enzyme activation achieved only by protein engineering, represents a step further in enhancing the enzyme's responsiveness. On the other hand, engineered β-galactosidases are also highly dynamic without preliminary antibody incubation, rendering activation factors around 300% after global reaction times shorter than 15 min. Therefore, this enzymatic system has been revealed as extremely robust and suitable for efficient and fast molecular detection in the diagnosis of infectious diseases.