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| Explore the Structural Domains Of CENP-E Protein Interacting with Mps1 protein by FRET method |
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Abstract Objective: To explore the structural domains of the CENP-E protein that interact with Mps1 protein. Methods: Two recombinant vectors named pEGFP- CENPE2 (containing 674-1085 amino acids of CENP-E protein) and pEGFP-CENPE 3(containing 1200~2134 amino acids of CENP-E protein) were transfected into human embryo kidney 293 (HEK293) cells respectively. The respective energy transfer efficiency (Ef) between either EGFP-CENPE2 and Mps1, or EGFP-CENPE3 and Mps1 were detected by FRET through selective photobleaching of the acceptors. Results: Both recombinant proteins expressed in HEK293 cells transfected by the recombinant plasmids were found to co-localize with the Mps1 protein as confirmed by confocal microscopy. The Ef between EGFP-CENPE3 and Mps1 protein was ［(12.63±0.48)%, n=30］ and that between EGFP-CENPE3 and Mps1 protein was ［(3.17±0.21)%, n=30］ as revealed by the results from FRET，the result of FRET was confirmed by coImmunoprecipitate(CO-IP) method. When compared with that between the control and Mps1, the Ef between EGFP-CENPE3 and Mps1 was significantly higher（p<0.05）, The results of CO-I Pshowed that EGFP-CENPE3 could interact with Mps1 protein. Conclusion: There was interaction between EGFP-CENPE3 and Mps1 as was demonstrated by the methods of FRET and CO-IP.
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Received: 29 December 2008
Published: 27 April 2009
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