Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritimaT = 1 and Myxococcus xanthusT = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthusT = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritimaT = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritimaT = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

Table S2: List of strains and plasmids used in this study.

Name Description Source
C321 ΔA exp 'Amberless' E. coli strain, all genomic UAG Amber codons replaced with UAA and the Amber associated release factor (RF1) gene deleted.For Non-canonical Amino Acid (NcAA) incorporation.

Figure S2 :
Figure S2: In vitro sfGFP loading is cargo loading peptide dependent.Molar ratio of sfGFP to encapsulin monomer 0.2, 1, 5 and 10 to 1 respectively.M=molecular weight marker.Top black and white image shows fluorescence signal of sfGFP.Bottom image shows Coomassie stained BN-PAGE gel.A: sfGFP loading into Tm_encap: No visible signal for sfGFP in fluorescence image, whereas fluorescence bands are visible with TmCLP and weak signal with MxCLP at 10:1.B: sfGFP loading into Mx_encap: No visible signal for sfGFP in fluorescence image, whereas fluorescence bands are visible with MxCLP and weak signal with TmCLP at 10:1.

Figure S3 :
Figure S3: sfGFP cargo loading and scaffolding effect.sfGFP cargo loading into Tm_encap (disassembled in 0.15 M NaOH) and Mx_encap (disassembled in 8 M urea) at increasing concentration of sfGFP.Top black and white image shows fluorescence signal of sfGFP, bottom image shows Coomassie stained BN-PAGE gel.M=molecular weight marker, A=assembled (before denaturation), vivo=in vivo loaded encapsulins, numbers in lanes indicate molar ratio of sfGFP to encapsulin monomer.Asterisk indicates intermediate species between T=1 and T=3 capsids.

Figure S4 :
Figure S4: Size Exclusion Chromatography (SEC) profiles of sfGFP in vitro loading.A-B: B is a zoom-in on A. Tm_encap in vivo assembled and purified capsid (dashed line), following 0.15 M NaOH disassembly (black), reassembled in the absence of sfGFP (dark blue) and in the presence of sfGFP cargo at 5:1 molar ratio with TmCLP (light blue), MxCLP (red) and without CLP (yellow).C-D: D is a zoom-in on C. Mx_encap in vivo assembled and purified capsid (dashed line), following 8M Urea disassembly (black), assembled in the absence of sfGFP (purple), reassembly in the presence of sfGFP cargo at 5:1 molar ratio with TmCLP (light blue), MxCLP (red) and without CLP (yellow).

Figure S5 :
Figure S5: SEC profiles of transketolase in vitro loading.A-B: B is a zoom-in on A. Tm_encap reassembly with TK cargo at a 1:1 molar ratio (following 0.15 M NaOH disassembly).Tm_encap in vivo assembled and purified (dashed line), reassembled in the presence of TK-TmCLP (light blue), TK-MxCLP (red) and sfGFP-TmCLP as a reference (light grey dot-dash).

Figure S6 :
Figure S6: Dynamic Light Scatter (DLS) size frequency distributions by volume of reassembled Tm_encap.A: Tm_encap in vivo assembled and purified capsid (dashed line), reassembled following 0.15 M NaOH treatment in the absence of cargo (blue) and in the presence of TK-TmCLP cargo (orange) at 1:1 molar ratio.B: Tm_encap loading with TK with different CLPs.Tm_encap in vivo assembled and purified (dashed line), reassembled following 0.15 M NaOH treatment in the presence of TK without CLP (green), with TK-TmCLP (orange), TK-MxCLP (purple).

Figure S7 :
Figure S7: Expression TK-TmCLP in BL21Star (DE3) and C321 ΔA E. coli for pAzF incorporation.A: Anti-His Western blot of expression of TK-TmCLP K603pAzF in BL21 Star (DE3) and C321 strains.W=whole cell lysate, I=insoluble fraction and S=soluble fraction of lysate.TK=Transketolase without TmCLP and pAzF as control.Note, the anti-His antibody did not detect TK well.B: SDS-PAGE of purification of TK-TmCLP K603pAzF in BL21 Star (DE3) and C321 strains.S=Soluble lysate and IMAC fractions at increasing imidazole concentrations (mM), elution at 500 mM and 5fold concentrated sample (con) of 500 mM elution fraction.TK=TK-TmCLP without pAzF as control.

Figure S9 :
Figure S9: Excitation and emission 3D scan of Tm_encap at 0.4 mg/mL.Contour levels indicate Counts per Second (CPS) ranges given in the heat scale.Grey indicates incident light detection.The black drop lines indicate excitation and emission peak of FRET Donor AlexaFluor 488.

Table S1 :
Primers used for the addition of cargo loading peptides and site directed mutagenesis (SDM) of TK-TmCLP K603 residue.