Recombinant Expression, Purification, and Assembly of p62 Filaments
This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.
Key wordsp62 Autophagy Filaments Affinity chromatography Negative stain electron microscopy
We acknowledge the assistance by the EMBL’s Protein Expression and Purification Core Facility for reagents and Electron Microscopy Core Facility for the instrument support.
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