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Microfluidic protein isolation and sample preparation for high-resolution cryo-EM
Journal
Proceedings of the National Academy of Sciences of the United States of America
Volume
116
Number
30
Pages / Article-Number
15007-15012
Keywords
cryo-EM; endogenous; microfluidics; protein purification; sample preparation
Mesh terms
Biotinylation; 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, ultrastructure; Protein Conformation; Protein Subunits, isolation & purification; Vitrification
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.
