Programmed loading and rapid purification of engineered bacterial microcompartment shells - PubMed (original) (raw)

Programmed loading and rapid purification of engineered bacterial microcompartment shells

Andrew Hagen et al. Nat Commun. 2018.

Abstract

Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Comparison of different shell preparation methods. a SDS-PAGE analysis of HO shell preparations. Lane 1: Classic, Lane 2: in vivo capped, Lane 3: ex vivo capped. Shell protein identities indicated by arrows; loading normalized to A280 readings. b–d Negative stain TEM micrographs of three different shell preparations and corresponding structural models. Scale bar = 100 nm. e SDS-PAGE analysis of eluates from pentamer titration experiment. Lane 1: Ex vivo capped shells from Fig. 1a, Lanes 2–6: Titration of pentamers with equivalent volumes of each culture used in mixing experiments expressed in milliliters. f SDS-PAGE analysis of Halo carboxysome shell preparation using affinity purification. g Negative stain TEM micrographs of Halo carboxysome shell preparation. Scale bar = 50 nm. Results presented are representative of two independent biological replicates for a–d, f, and g. Findings in figure (e) recapitulate pilot experiments in which a subset of specified lysate ratios were used

Fig. 2

Fig. 2

Comparison of different minimal shells. a SDS-PAGE analysis of crude shell preparations. Lane 1: HT1PSII Lane 2: HT2PSII Lane 3: HT3PSII. Shell protein identities indicated by arrows. b–d Negative stain TEM micrographs of three minimal shell preparations and corresponding structural models. Scale bar = 100 nm

Fig. 3

Fig. 3

Molecular models of T1, TSC and TST subunits. a Model of T1 (wt) (PDB: 5DIH) as viewed from side of shell (top) and lumen (bottom). b and c Models of TSC and TST (respectively) viewed from the side. Flexible coil regions colored in brown; SpyCatcher and SpyTag regions colored in gray

Fig. 4

Fig. 4

SDS-PAGE and electron micrographs of various shell preparations. a, b SDS-PAGE of shell preparations. Composition of shell preparations given in tabular form below each lane. c Negative stain TEM of HTSC~STcfpPSII shell preparation (lane 6 in figure a) and cutaway model of shells (cfp rendered in turquoise, not completely functionalized for clarity). Results presented are representative of at least two independent biological replicates of each sample preparation

Fig. 5

Fig. 5

Fluorescence spectra and SDS-PAGE analysis of shells containing ex vivo programmed cargo. a Scaled emission spectra (excitation: 405 nm) of programmed cargo. STyfp-only trace (0:10) was not plotted—in the absence of a FRET donor, the fluorescence signal is negligible. b SDS-PAGE analysis of STcfp (10:0) programmed shells. c SDS-PAGE analysis of SCcfp programmed shells. Results are representative of two independent biological replicates

Fig. 6

Fig. 6

Probe properties and permeability assay of uncapped and capped shells. a Schematic of probe behavior in presence of FlAsH and TEV protease. b Fluorescence emission spectra (excitation: 405 nm) of unencapsulated probe. c Emission spectra (450–600 nm) of encapsulated probe in uncapped and capped shells, in the presence of FlAsH and TEV protease. Axis numbers omitted for clarity; tick marks correspond to numbers in b. Results are representative of two independent technical replicates. Bacterial microcompartments are protein-bound organelles encapsulating segments of metabolic pathways. Here the authors functionalize shell proteins to facilitate facile purification and enable cargo encapsulation via covalent linkage

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