Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

Proc Natl Acad Sci U S A. 2019 Jul 23;116(30):15007-15012. doi: 10.1073/pnas.1907214116. Epub 2019 Jul 10.

Abstract

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.

Keywords: cryo-EM; endogenous; microfluidics; protein purification; sample preparation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • Cryoelectron Microscopy / instrumentation
  • Cryoelectron Microscopy / methods*
  • HeLa Cells
  • Humans
  • Image Processing, Computer-Assisted / statistics & numerical data*
  • Imaging, Three-Dimensional
  • Immunoglobulin Fab Fragments / chemistry
  • Microfluidic Analytical Techniques / methods
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / isolation & purification
  • Proteasome Endopeptidase Complex / ultrastructure*
  • Protein Conformation
  • Protein Subunits / chemistry*
  • Protein Subunits / isolation & purification
  • Vitrification

Substances

  • Immunoglobulin Fab Fragments
  • Protein Subunits
  • Proteasome Endopeptidase Complex