PROTEIN INTERACTIONS: HIGH THROUGHPUT SUBCLONING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Further development and implementation of methods for high throughput (HT) subcloning. The ability to subclone many ORFs into different vectors is increasingly important to many proteomic analyses. Subcloning is essential, for example, for high throughput protein interaction assays including those based on the yeast two-hybrid system and protein microarrays. Subcloning is also necessary for determination of protein complexes by mass spectrometry, which requires expression of tagged proteins. It is also essential for structural determinations, which require purified proteins. We will further develop three approaches to high-throughput subcloning. One uses recombination in yeast, which is particularly well suited for constructing yeast expression clones for assays like the yeast two-hybrid system. The second approach uses recombination in E. coli, and the third uses a modified version of the GatewayTM in vitro cloning system developed by Invitrogen.