Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

Abstract

A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

“…Although most of the previous and current studies were carried out using recombinant proteins and protein complexes due to its easy accessibility and less complexity, it is expected that the study of protein complexes in a natural context, such as in living cells, will boost in the near future. One of the main challenges in characterizing endogenous complexes is their low abundance, advances in enrichment techniques including biochemical purification, immunoprecipitation, and affinity tagging are crucial for enabling application of nTDMS to less abundant proteins (LaCava et al, 2015; Shiraiwa et al, 2020; Zhao et al, 2021). Cheng et al demonstrated that by combining CRISPR/Cas gene editing and fluorescence cell sorting, rapidly tag and purify endogenous human proteins from cell lines were achievable, enabling structural analysis of native proteins that are properly folded and assembled using cryoEM (Zhao et al, 2021).…”

“…One of the main challenges in characterizing endogenous complexes is their low abundance, advances in enrichment techniques including biochemical purification, immunoprecipitation, and affinity tagging are crucial for enabling application of nTDMS to less abundant proteins (LaCava et al, 2015; Shiraiwa et al, 2020; Zhao et al, 2021). Cheng et al demonstrated that by combining CRISPR/Cas gene editing and fluorescence cell sorting, rapidly tag and purify endogenous human proteins from cell lines were achievable, enabling structural analysis of native proteins that are properly folded and assembled using cryoEM (Zhao et al, 2021). Advances in mass spectrometer technology are also increasing the sensitivity of measurements and enabling application of biological MS to less abundant proteins, such as the recently released timsTOF SCP (Bruker) that is a dedicated ultrahigh sensitivity instrument for unbiased single cell proteomics (Brunner et al, 2022).…”

The Proteasome Activator PA200/PSME4: An Emerging New Player in Health and Disease

“…PA200 was also previously reported to form PA200-20S-19S proteasome complexes upon irradiation in HeLa cells [ 21 ]. A recent BioRxiv paper also captured the structures of PA200-20S-PA28, as well as the PA200-20S-19S complexes [ 22 ]. The cross-linking proteomics analysis from the lab of Marie-Pierre Bousquet confirmed the presence of PA200 as part of the 20S, but also of the 26S complexes.…”