title: Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin.
abstract: The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble in vivo. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from Thermotoga maritima both in vivo and in vitro by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a simple and rapid purification protocol and demonstrate the thermal and pH stability of the shell. Efforts to study the biophysical effects of protein encapsulation have been problematic in complex compartments, but the simplicity of assembling and loading encapsulin makes it an ideal system for future experiments exploring the effects of encapsulation on proteins.
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Caleb
authors.initials: C
authors.lastname: Cassidy-Amstutz
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Luke
authors.initials: L
authors.lastname: Oltrogge
authors.affiliation: Department of Chemistry, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Going
authors.affiliation: Department of Physics, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Antony
authors.initials: A
authors.lastname: Lee
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Poh
authors.initials: P
authors.lastname: Teng
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: David
authors.initials: D
authors.lastname: Quintanilla
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Alexandra
authors.initials: A
authors.lastname: East-Seletsky
authors.affiliation: Department of Chemistry, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: Evan R
authors.initials: ER
authors.lastname: Williams
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley , Berkeley, California 94720, United States.|Department of Chemistry, University of California at Berkeley , Berkeley, California 94720, United States.|Energy Biosciences Institute, University of California at Berkeley , Berkeley, California 94720, United States.
authors.forename: David F
authors.initials: DF
authors.lastname: Savage
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D066298:In Vitro Techniques; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D020124:Thermotoga maritima / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010544:Peroxidases