PORTEFEUILLE

Protein microarrays and microchips are becoming increasingly important tools for large scale and high-throughput protein detection and screening of protein function. Despite their successful development and implementation, current microarray and microchip technology still suffers from some drawbacks, which significantly limit sensitivity, reliability and reproducibility. In this project, a recently developed, unique method for covalent protein modification was combined with advanced, surface-initiated controlled radical polymerization techniques to overcome some of the limitations of current protein microarray and microchip technology. The goal was to create a technology platform, which could serve as the basis for the development of a prototype, next generation protein microarray/microchip in a more application-oriented follow-up project.

The pre-competitive, application-oriented research that was carried out resulted in a technology platform that may be used in a follow-up project for the development of prototype devices. The project had strong interdisciplinary character and combined aspects from materials science, polymer chemistry, biological chemistry and biotechnology. The project was carried out as a joint effort of two laboratories, which are part of two different schools and institutes at EPFL (Institute of Materials/School of Engineering Sciences and Technology and Institute of Chemical Sciences and Engineering/School of Basic Sciences). The different, but complementary backgrounds of the applicants generated synergies, which were very beneficial to the project.

One of the project partners recently developed a general method for covalent protein labelling, which involves the use of O6-alkylguanine-DNA-alkyltransferase (AGT) fusion proteins and relies on the ability of AGT to irreversibly transfer the alkyl-residue from O6-benzylguanine derivatives. The AGT mediated functionalization offers some unique advantages: (1) functionalization occurs exclusively via the AGT fusion, leaving the protein of interest accessible for interactions with other molecules; (2) functionalization is covalent; (3) functionalization is very chemoselective and can be carried out both in vivo and with crude cell lysates without the need for purification steps; (4) the substrate specificity of AGT is relatively low and allows the attachment of a broad range of chemical and spectroscopic probes.

Surface properties are critical to bioanalytical applications. A surface is needed that allows covalent immobilization of the protein of interest and also prevents non-specific adhesion of the analyte. Thin polymer layers are widely used to modify surface properties. Two general methods can be distinguished for the preparation of thin polymer coatings; (i) the grafting of polymer chains onto an activated surface (“grafting-onto”); (ii) the polymerization of monomers from a surface (“grafting-from”). The advent of controlled radical polymerization techniques such as nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT) over the past decade makes the “grafting-from” approach particularly attractive.

In this project, the AGT mediated covalent protein immobilization and surface-initiated controlled radical polymerization were combined to develop a new generation of protein microarrays. Thin polymer layers (brushes) have been prepared that show significantly enhanced resistance towards non-specific protein adhesion compared to glass and polystyrene surfaces. To this end, polymer brushes were prepared from three different ethylene glycol based methacrylate macromonomers and the resulting surfaces characterized with regards to non-specific protein adhesion. It could be shown that these polymer brushes can be used as platforms to anchor benzylguanine moieties and subsequently immobilize O6-alkylguanine DNA-alkyltransferase fusion proteins. Fusion proteins immobilized following this strategy have been used to screen protein – protein and protein – small molecule interactions and to detect posttranslational modifications. These results indicate that the functional properties of the proteins are not affected by the immobilization procedure. In a further series of experiments, the inhibitor binding activity of an enzyme that was immobilized via the AGT strategy was compared with that of an enzyme which was immobilized on a commercially available, aldehyde functionalized chip. These experiments revealed that the activity of the AGT immobilized enzyme towards binding of the inhibitor was approximately two times higher compared to the enzyme immobilized on the commercially available chips. These results clearly underline the importance of surface engineering and immobilization chemistry for the development of protein microchips. As a final proof of concept experiment, it was planned to fabricate a microarray with a set of 35 proteins from Mycobacterium tuberculosis. The corresponding AGT fusion proteins were expressed both in E. coli and M. smegmatis. Unfortunately, these proteins were found to degrade rapidly. Since degradation products that carry the AGT tag would compete with the full-length protein, no experiments were performed to immobilize these proteins on the polymer brushes. In the last part of the project, however, the polymer brushes were successfully used to generate microarray surfaces that can be activated with UV light to allow chemoselective binding of AGT fusion proteins. This further expands the potential use of the polymer brush based microarray coatings with regards to photopatterning and allows enhanced control over the spatial localization of proteins on the surface.

S. Tugulu, A. Arnold, I. Sielaff, K. Johnsson, H.-A. Klok, “Protein functionalized polymer brushes”, Biomacromolecules 2005, 6, 1602.
I. Sielaff, A. Arnold, G. Godin, S. Tugulu, H.-A. Klok, K. Johnsson, Protein function microarrays based on self-immobilizing and self-labelling fusion proteins, ChemBioChem 2006, 7, 194.
A. Arnold, S. Tugulu, K. Johnsson, H.-A. Klok, manuscript in preparation.
S. Banala, A. Arnold, K. Johnsson, ChemBioChem, submitted

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