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Isolation and Functional Studies of Eukaryotic Ribosomes using HaloTag Surface Display and Imaging Technology

The understanding of ribosome complex assembly, structure, and function has exploded in recent years by achievements in the areas of single molecule studies, electron microscopy, and X‐ray crystallography. To facilitate further biochemical and cellular characterization, we have developed a protein capture and labeling technology, HaloTag, and applied it for functional studies of ribosomes, including efficient isolation and covalent attachment to surfaces as well as fluorescent labeling to visualize in vivo localization. Using mass spectrometry analysis we show successful capture intact 80S ribosomes directly from a mammalian cellular lysates. A notable advantage for analysis with mass spectrometry is the feature that HaloTag remains bound to the resin during the elution step. Therefore the sample is not plagued with the capture molecule, allowing for a more efficient analysis of the sample and detection of dilute and small proteins. Further experiments were done to show that the isolated ribosomes were bound to mRNAs and retained activity in vitro for elongation and complete protein synthesis. Correlative pulse‐chase cellular imaging experiments were done to follow old and newly assembled ribosomal populations after starvation. The multiple capabilities of this technology demonstrate the ability to fully characterize ribosomal assembly, interactions, function, and cellular localization.