title: Directed evolution of a protein container.
abstract: Confinement of enzymes in protein nanocompartments represents a potentially powerful strategy for controlling catalytic activity in cells. By using a simple electrostatically based tagging system for protein encapsulation, we successfully sequestered HIV protease, a toxic enzyme when produced cytoplasmically, within an engineered lumazine synthase capsid. The growth advantage resulting from protecting the Escherichia coli host from the protease enabled directed evolution of improved capsids. After four rounds of mutagenesis and selection, we obtained a variant with a 5- to 10-fold higher loading capacity than the starting capsid, which permitted efficient growth even at high intracellular concentrations of HIV protease. The superior properties of the evolved capsid can be ascribed to multiple mutations that increase the net negative charge on its luminal surface and thereby enhance engineered Coulombic interactions between host and guest. Such structures could be used for diverse biotechnological applications in living cells.
authors.affiliation: Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland.
authors.forename: Bigna
authors.initials: B
authors.lastname: Wörsdörfer
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Woycechowsky
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000595:Amino Acid Sequence; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D016333:HIV Protease / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D015202:Protein Engineering; D012641:Selection, Genetic; D055672:Static Electricity; D014169:Transformation, Bacterial
chemical_list: D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D016333:HIV Protease