The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.

This paper describes a physical organic study of the relationship between the rate for an interfacial Diels-Alder reaction and the steric environment around the reacting molecules. The study used as a model reaction the cycloaddition of cyclopentadiene with a self-assembled monolayer (SAM) presenting benzoquinone groups surrounded by hydroxyl-terminated alkanethiolates. The accessibility of the quinone was varied by preparing monolayers from hydroquinone-terminated alkanethiols of different lengths [HS(CH(2))(n)-HQ, n = 6-14] and a hydroxyl-terminated alkanethiol [HS(CH(2))(11)-OH] of constant length. Cyclic voltammetry was used to measure the rate of the reaction by monitoring the decay of the redox-active quinone. The second-order rate constant showed a modest change as the position of quinone was varied relative to the hydroxyl groups of the monolayer. For monolayers wherein the quinone groups were extended from the interface, the rate constants oscillated near 0.20 M(-1) s(-1) with an even-odd dependence on the length of the alkanethiol. For monolayers that positioned the quinone groups below the surrounding hydroxyl groups, the rate constants decreased by approximately 8-fold. Examination of the activation parameters revealed that the quinone groups that were positioned below the interface (and in a crowded environment) reacted with an enthalpy of activation that was 4 kcal/mol greater than did the quinones that were accessible at the interface. The reaction of the buried quinone, however, proceeded with an entropy of activation that was more favorable by 13 eu, and therefore with a similar free energy of activation. The combination of SAMs for preparing model interfaces and cyclic voltammetry for measuring rates provides a new opportunity for physical organic studies of interfacial reactions.

Without a trace: The immobilization of biologically active proteins onto a solid support through split‐intein‐mediated protein trans‐splicing reactions requires only very dilute samples. Not only is this process traceless but it does not require purification of the protein, thereby making possible the direct immobilization of proteins from complex mixtures such as cell lysates.

Antibody arrays hold considerable potential in a variety of applications including proteomics research, drug discovery, and diagnostics. Many of the schemes used to fabricate the arrays fail to immobilize the antibodies at a uniform density or in a single orientation; consequently, the immobilized antibodies recognize their antigens with variable efficiency. This paper describes a strategy to immobilize antibodies in a single orientation, with a controlled density, using the covalent interaction between cutinase and its suicide substrate. Protein fusions between cutinase and five antibodies of three different types (scFv, V(HH), and FN3) were prepared and immobilized upon self-assembled monolayers (SAMs) presenting a phosphonate capture ligand. The immobilized antibodies exhibit high affinity and selectivity for their target antigens, as monitored by surface plasmon resonance and fluorescence scanning. Furthermore, by changing the density of capture ligand on the SAM the density of the immobilized antibody could be controlled. The monolayers, which also present a tri(ethylene glycol) group, are inert to nonspecific adsorption of proteins and allow the detection of a specific antigen in a complex mixture. The demonstration of cutinase-directed antibody immobilization with insert SAMs provides a straightforward and robust method for preparing antibody chips.