SynBERC:MIT/Calendar/2007-7-11

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Anyone in the synthetic biology community is welcome to attend.

Wednesday July 11, 2007 at 12:30pm EST

32-G449, MIT

Topic of discussion

Deborah F. Kelly from the Department of Cell Biology at Harvard Medical School will be speaking on some of her recent work.

Monolayer purification: A rapid method for isolating protein complexes for single particle Electron Microscopy

Deborah F. Kelly, Danijela Dukovski and Thomas Walz

Visualizing macromolecular complexes by cryo-electron microscopy (EM) usually entails the biochemical purification of the complex, followed by specimen vitrification on holey carbon grids, imaging using low-dose procedures and subsequent image processing to calculate a three-dimensional (3D) reconstruction. Here we introduce the use of lipid monolayers as a novel method to produce vitrified samples of macromolecular complexes without prior biochemical purification. The method can be used for His-tagged proteins and employs lipid monolayers that contain lipids with a modified Ni-NTA headgroup. To test the method we used a His-tagged construct of the transferrin (Tf)-transferrin receptor (TfR) complex. Various amounts of the Tf-TfR complex were added to mammalian and insect cell extracts, which were overlayed with a Ni-NTA-lipid monolayer. After transfer of the monolayer onto an EM grid, the specimens were visualized using negative staining. Nanogram quantities of the Tf-TfR complex in extracts of 3 –7 mg/ml protein concentration were sufficient to specifically adsorb only the Tf-TfR complex to the monolayer. The sensitivity could be further improved to purify as little as 10 ng of Tf-TfR complex from the same concentration of cell extracts upon the addition of imidazole to the subphase. We could further vitrify the monolayer purified Tf-TfR specimens and calculate a 3D reconstruction to a nominal resolution of 20 Å. Furthermore, the specificity of the method was ascertained by eluting the complexes from the monolayer followed by tandem mass spectrometry, which identified only the constituents that make up the his-tagged complex. We are currently using this approach to visualize other protein complexes obtained from commercially available libraries of human His-tagged constructs.

Acknowledgments

Thanks to Julie Norville for organizing this lunch.