Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays.

A novel strategy to modulate the assembly and trans-splicing activity of the Ssp DnaE split-intein was achieved by introducing two photolabile protecting groups onto the backbone of the C-intein polypeptide. This modification was not only able to efficiently block the trans-splicing activity, but also reduce significantly the binding affinity constant between the C-and N-intein fragments. The original activity of the wild-type split intein could be fully recovered by brief exposure to UV light. KeywordsBiotechnology; Proteins; Peptides; Photolysis; Recognition Intein-mediated protein splicing is a naturally occurring self-processing event in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond.[1-4] Protein splicing can be found to occur in cis or in trans. [5][6][7] In protein trans-splicing the intein self-processing domain is split in two fragments, called N-intein (I N ) and C-intein (I C ), respectively. These two intein fragments are inactive individually. However, they can bind each other with high specificity under appropriate conditions to form a functional protein-splicing domain.Since it was initially discovered, intein-mediated protein splicing and trans-splicing have been used in a multitude of applications ranging from protein purification, [8] protein backbone cyclization,[9-12] addition of labeled tags or other moieties, [13,14] protein immobilization, [15][16][17][18] protein semi-synthesis, [7] and segmental labeling or modification. [19][20][21][22] Of particular interest is the use of conditional protein trans-splicing, [23,24] which allows controlling the activity of a particular protein by modulating the assembly of the split intein moieties and therefore its splicing activity (Fig. 1).Recent work by Muir and co-workers has shown that the use of a reversible O-acyl backbone modification on the Ssp DnaE I C can be used to modify the splicing activity of this split-intein.[25] However, the lack of trans-splicing activity associated with the different O-acyl C-intein analogues was shown to be due to the introduction of local perturbations in the active site of

This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function.