In-Cell Engineering of Protein Crystals with Nanoporous Structures for Promoting Cascade Reactions

Abstract

The development of solid biocatalysts has rapidly progressed for applications in nanomaterials. The immobilization of enzymes in solid materials is considered an alternative method for constructing traditional biocatalysts with high stability and efficiency to be reused. However, the design of solid scaffolds from nanoporous materials to immobilize multiple enzymes and improve catalytic reactivity remains challenging. In this study, the engineering of in-cell protein crystals is demonstrated to have the potential for immobilizing two enzymes that promote a cascade reaction. Polyhedra is known as an in-cell crystalline protein produced in insect cells by the infection of cytoplasmic polyhedrosis virus. We constructed the 38-residue-deletion mutant, which forms interlinked hollow nanocages with a diameter of 5 nm in the crystal. The mutant crystal can encapsulate Candida antarctica lipase B and Lactobacillus kefir alcohol dehydrogenase. The composite crystal significantly enhances the reactivity of the cascade reaction with 1.9-fold and 3.8-fold higher efficiency than that of the wild-type crystal and the mixture of the free enzymes, respectively. The higher reactivity is because of the substrate and intermediate efficiently diffusing through the extended channels designed within the nanoporous crystal. These results suggest the possibility of using polyhedrin crystals to design the nanoporous materials for developing further applications in bio-nanomaterial science.

Segregated Protein–Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons

“…Protein-based crystalline materials are advancing in sophistication, [6][7][8][9][10][11][12][13][14][15] including in vivo construction [6,7,11,13] and industrial application, [11,13] although reliable self-assembling systems are scarce. Tried-and-tested systems such as ferritin [8] and polyhedrin [7,11] are the main focus while ligand-mediated assembly continues to deliver remarkable bionanoarchitectures with the potential for customization. [8][9][10]15,[20][21][22] RSL, with its high rigidity and symmetry, has been used extensively in bio-supramolecular chemistry.…”

“…Protein‐based crystalline materials are advancing in sophistication,[ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] including in vivo construction[ 6 , 7 , 11 , 13 ] and industrial application,[ 11 , 13 ] although reliable self‐assembling systems are scarce. Tried‐and‐tested systems such as ferritin [8] and polyhedrin[ 7 , 11 ] are the main focus while ligand‐mediated assembly continues to deliver remarkable bionanoarchitectures with the potential for customization.…”

“…Larger industrial enzymes have been accommodated in crystals of engineered polyhedrin. [11] The RSL*-Q7 sheet architecture can also be used as a scaffold to accommodate fused protein subunits, as evidenced with Mefp-RSL* and 4dzn-RSL*. Whereas other systems have relied on insertion of fusions into large solvent voids, [12][13][14] here, the N-terminal fusions are wedged in between RSL*-Q7 bilayers (Figures 3 and 5).…”

“…Engineered protein crystals[ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ] provide a basis for functional biomaterials with applications such as templated catalysis [11] and protein encapsulation. [ 12 , 13 , 14 ] Different engineering approaches currently studied include deletion mutations to generate cavities,[ 7 , 11 ] metal‐directed protein–protein interfaces, [8] as well as naturally porous (fusion‐)protein crystals. [ 12 , 13 , 14 ] Ligand‐mediated assembly is another route to tuneable protein architectures.…”

Time-tagged ticker tapes for intracellular recordings

“…We considered many possibilities, including endogenous microtubules, bacterial R-bodies, 10 plant forisomes, 11 amyloid fibrils, 12 prions, 13 filamentous viruses, 14 crystals of the fluorescent protein XpA, 15 engineered fiber-forming peptides, 16 and other proteins that spontaneously crystallize in cells. 17 A fusion of the catalytic domain of the Pak4 kinase and its inhibitor Inka1 (hence called iPAK4) largely satisfied the selection criteria. This construct has been shown to stably assemble in cells into rod-shaped crystals.…”