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'Microarray' might just be the hottest buzzword in biology today. Small molecules, oligonucleotides and proteins are being immobilized onto surfaces for various high throughput screening studies. There are unique chemical challenges in the immobilization of each type of molecule onto a surface, yet as the technology advances, thankfully there are more and more available solutions.

Protein immobilization on a microarray surface is especially tricky because of the inherent sensitivity of proteins to perturbations. “The display of proteins in microarray format is a problem for which there is no general solution yet,” remarks Caltech professor David Tirrell. Initial efforts to construct protein arrays exploited the inherent reactivity of lysine or cysteine residues to covalently capture proteins with a reactive group on the surface, but this can cause active sites to become unavailable, or it can even prompt denaturation of the protein. Many researchers since have borrowed traditional affinity chromatography methods to display proteins on surfaces, such as fusing the protein of interest with a hexahistidine tag for capture by immobilized metal or biotinylating the protein for capture by avidin/streptavidin. The challenge still remains, however, to construct tags that are extremely stable under a variety of conditions, while maintaining high affinity for the surface chemistry. Toward that goal, Tirrell and colleagues recently introduced a new flexible polypeptide scaffold consisting of a surface immobilization domain and a protein capture domain.

The immobilization domain is composed of a highly hydrophobic elastin mimetic peptide that strongly adheres to hydrophobic surfaces. Furthermore, incorporation of an unnatural photoreactive phenylalanine derivative allows the scaffold to be covalently linked to an appropriately derivatized surface upon UV irradiation. The protein capture domain consists of one helix of a leucine zipper coiled coil. The complementary helix is expressed as a fusion with the target protein of interest. Owing to the extremely tight and specific noncovalent association of the leucine zipper coiled coil, the protein of interest can be efficiently captured by the immobilized scaffold (Fig. 1), even from a crude cell lysate.

Figure 1: A new polypeptide scaffold for flexible protein arraying consists of an elastin mimetic domain and a leucine zipper helix.
figure 1

The elastin mimetic domain is covalently bound to the surface by a photoreactive amino acid. The target protein is fused to the complementary leucine zipper helix; strong noncovalent association of the helices leads to dimerization, effectively immobilizing the target protein.

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The engineered polypeptide scaffold allows much greater flexibility in the immobilization of proteins on a microarray for functional studies. “Because proteins have such variable chemistries, it is difficult to array diverse sets of proteins,” explains Tirrell. “We are able to link proteins to a surface, but we are also able to provide some diversity in the nature of the linkage by using protein design”.