Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

Abstract

Protein arrays are ideal tools for the rapid analysis of whole proteomes as well as for the development of reliable and cheap biosensors. The objective of this proposal is to develop a new ligand assisted ligation method based in the naturally occurring protein trans-splicing process. This method has been used for the generation of spatially addressable arrays of multiple protein components by standard micro-lithographic techniques. Key to our approach is the use of the protein trans-splicing process. This naturally occurring process allows the development of a truly generic and highly efficient method for the covalent attachment of proteins through its C-terminus to any solid support. This technology has been used for the creation of protein chips containing several virulence factors from the human pathogen Y. pestis. IntroductionMany experimental techniques in biology and biophysics, and applications in diagnosis and drug discovery, require proteins immobilized on solid substrates. [1][2][3] In fact, the concept of arrays of proteins attached to a solid support has attracted increasing attention over the last three years due to the sequencing of several genomes, including the human genome. When a genome has been deciphered, the daunting task of determining the function of each protein encoded in the genome still remains. Protein arrays can be used easily for such analysis in a parallel fashion.2,4 Another powerful application employs ordered nanometric arrays of proteins as nucleation templates for protein crystallization or for structural studies. Recent advances in nanoprinting techniques have allowed the creation of sub-micrometer arrays of proteins. 5,6 All these applications demonstrate the use of protein arrays and also highlight the need for methods able to attach proteins in a well defined and ordered way onto a solid supports.Various methods are available for attaching proteins to solid surfaces. Most rely on non-specific adsorption, 6,7 or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with surfaces containing complementary reactive groups. 8,9 In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue. However, in most cases the interactions of the tags are reversible and therefore not stable over the course of subsequent assays or require large mediator proteins.10,11 Methods for the chemoselective attachment of proteins to surfaces has been also developed recently by our group.12-15 These methods rely in the introduction of two unique and mutually reactive groups on the protein and the support surface. The reaction between these two groups usually gives rise to the selective attachment of the protein to the surface with total control over the orientation.14,15 However, these methods, although highly selective, rely on uncatalyzed pseudo-bimolecular reactions with little or not activation at all. This lack of activation means that the efficiency of these bim...