Site-specific and effective immobilization of proteins by Npu DnaE split-intein mediated protein trans-splicing reaction

Abstract

Immobilized proteins on solid supports provide a useful platform for various biological assays including proteomics research, protein-protein interaction studies, and diagnosis. In fabricating protein chips, proteins should be immobilized to maintain activity and specificity towards their targets. Here, we describe an approach to attach a protein of interest onto a solid support through a covalent bond as well as in situ monitoring of protein immobilization and subsequent binding assays. Our system utilized self-assembled monolayers of alkanethiolates on gold as biologically inert solid substrates, and Nostoc punctiforme DnaE splitinteins were used to mediate biospecific interactions and covalently conjugate proteins on the solid support. Use of a gold substrate enabled in situ monitoring of the protein-protein interactions using surface plasmon resonance spectroscopy. This approach provides a flexible method for further protein immobilization applications.