H-066. High Throughput Mapping of Caulobacter crescentus Protein Interactions Using Tandem Affinity Purification

P. Hu1, J. Zhu1, G. L. Andersen1, L. Shapiro2, H. H. McAdams2, T. Earnest1;
1Lawrence Berkeley Natl. Lab., Berkeley, CA, 2Stanford Univ., Stanford, CA.

Background: Tandem Affinity Purification (TAP) has proven to be a powerful tool in the investigation of protein-protein interactions in yeast and in E. coli as it can purify stable multi-protein complexes in near native conditions. Regulation of Caulobacter crescentus cell division and the establishment of cell polarity involve many dynamic protein-protein interactions. This study has developed a high throughput system to purify multiple protein complexes from Caulobacter crescentus using TAP tag (consists of two IgG binding domain of Staphylococcus aureus protein A and a calmodulin binding peptide separated by a TEV protease cleavage site). Method: To ensure proper regulation in critical cellular processes, it is preferred to have in-frame fusion of the target protein to the TAP tag and the integration of the construct onto chromosomal loci. Special vectors need to be developed to accomplish such tasks in Caulobacter crescentus. Results: We have developed methods of integrating the TAP tag into the target ORF at the C-terminal using the Gateway system to allow for higher target throughput. Tagging of the N-terminal can be achieved by a more complicated protocol for targets of high scientific value where C-terminal tagging is not possible or when the C-terminal is cleaved during proteolytic regulation. In our initial test set, a wide range of targets covering a distribution of sizes and functionality, approximately 75% of the targets could be C-terminal TAP tagged and purified. Investigations of ORFs targeted due to their importance in cell polarity, cell division, and cell cycle control also exhibited a high success rate. Conclusion: A high throughput system has been developed to introduce TAP tagged target proteins in Caulobacter crescentus. It has been successfully tested with a variety of protein targets. Such system is an essential tool in identification of components in any biological complexes without prior knowledge of complex composition or function.