Each stage in the building of a protein chip — expression, purification, immobilization — adds a layer of experimental complexity, as each feature may need its own optimization process to ensure consistent quality. “We thought we needed a better way to do this,” says Joshua LaBaer, director of the Harvard Institute of Proteomics.

Joshua LaBaer uses self-assembling arrays to bypass problems with proteins.

The solution that he and his team arrived at was the 'nucleic acid programmable protein array' (NAPPA), in which cDNAs encoding GST fusion proteins are arrayed on chips alongside antibodies that recognize GST. The array is then subjected to cell-free transcription and translation; as protein is produced, it gets bound by an antibody and presented for analysis. According to LaBaer, NAPPA has simplified his group's research. “You don't have to purify proteins — you just purify DNA, so it's pretty easy, and it's been successful for printing about 95% to 96% of the things we make,” he says. “And when we do protein–protein interactions, we're getting interactions that make sense and not a lot of false positives.

Although initial arrays were limited in size, LaBaer and his team have since generated NAPPA arrays with up to 2,000 features, and they hope to surpass this soon.

Other techniques even bypass DNA immobilization. In the protein in situ array (PISA) developed by Michael Taussig of the Babraham Institute in Cambridge, UK, cDNAs are amplified in situ with primers that encode polyhistidine tags, so that proteins can be captured on a nickel–NTA-coated surface.

More recently, Philipp Angenendt of the German Cancer Research Center in Heidelberg transferred the PISA principle to a microarray set-up, integrating cell-free production of histidine-tagged protein from unpurified PCR fragments with a multiple spotting technique (MIST) previously developed by his group. MIST uses automation to apply array reagents precisely and sequentially to specific spots. As a result, each transcription/translation reaction is confined to a tiny, sub-nanolitre droplet, allowing greater density — up to 13,000 spots at present. “The nice thing about it is that the proteins expressed remain in a liquid environment,” says Angenendt, “and the structure should be as intact as it can be with a solid-phase immunoassay format.”

Both Angenendt and LaBaer are now fine-tuning their processes. “We're really working on large-scale screens, doing biomarker and protein interaction studies,” says LaBaer. “We've also got some preliminary enzymatic data that look promising.”

Michael Eisentein