In vitro protein microarrays for detecting protein‐protein interactions: Application of a new method for fluorescence labeling of proteins

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Abstract
Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein‐protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell‐free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore‐puromycin conjugate was incorporated into a protein at the C‐terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein‐protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high‐throughput format and also combined with in vitro display technologies such as the streptavidin‐biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227–230), our method should be useful for large‐scale analysis of protein‐protein interactions.