Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
pubs.acs.org/Biomac
Multifunctional Enzyme Packaging and Catalysis in the Qβ Protein Nanoparticle Jason D. Fiedler,§ Maxwell R. Fishman,§ Steven D. Brown,§ Jolene Lau,§ and M. G. Finn*,§,‡ §
Biomacromolecules Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/30/18. For personal use only.
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States ‡ School of Chemistry and Biochemistry, School of Biological Sciences, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, United States S Supporting Information *
ABSTRACT: The simultaneous expression in Escherichia coli cells of the Qβ virus-like particle (VLP) capsid protein and protein “cargo” tagged with a positively charged Rev peptide sequence leads to the spontaneous self-assembly of VLPs with multiple copies of the cargo inside. We report the packaging of four new enzymes with potential applications in medicine and chemical manufacturing. The captured enzymes are active while inside the nanoparticle shell and are protected from environmental conditions that lead to free-enzyme destruction. We also describe genetic modifications to the packaging scheme that shed light on the self-assembly mechanism of this system and allow indirect control over the internal packaging density of cargo. The technology was extended to create, via self-assembly, VLPs that simultaneously display protein ligands on the exterior and contain enzymes within. Inverse relationships were observed between the size of both the packaged and externally displayed protein or domains and nanoparticle yield. These results provide a general method for the rapid creation of robust protein nanoparticles with desired catalytic and targeting functionalities.
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INTRODUCTION
The multi-component nature of the Qβ VLP packaging system lends itself to cargo flexibility and relative ease of use. We illustrate this here by the packaging of four different cargo enzymes with industrial or therapeutic relevance: (a) 2deoxyribose-5-phosphate aldolase (E.C. 4.1.2.4, DERA), important for the biocatalytic synthesis of a variety of highvalue small molecules including statin drugs;46 (b) superoxide dismutase (E.C. 1.15.1.1, SOD),47 of interest as a localized anti-inflammatory agent (Orgotein);48−50 (c) cytosine deaminase (E.C.3.5.4.1, CD),51,52 which converts orally available prodrug 5-fluorocytosine (5-FC) into the cytotoxic antimetabolite 5-fluorouracil (5-FU);53,54 and (d) purine-nucleoside phosphorylase (E.C. 2.4.2.1, PNP),55,56 an alternative prodrugmodifying enzyme that acts on nucleoside analogues to release toxic purine analogs 2-fluoradenine (F-Ade)57 and 6methylpurine.58 These enzymes are packaged into Qβ VLPs, are active while inside the shell, and are protected from degradation. In addition, we have also developed a method to display heterologous peptide domains on the exterior of the VLP shell with a co-expression system59 and show here that these two methods can be combined to create nanoparticles that package cargo enzymes and display targeting domains on the particle exterior. This self-assembling system requires only that
Enzymes are used in large quantities in many diverse applications.1−6 Unfortunately, many of these catalysts do not tolerate the conditions that the intended applications require, 7 driving the use of extremophilic variants, 8,9 immobilized enzymes,10,11 or modified catalysts by directed evolution.12−14 The entrainment of heterologous enzymes inside protein shells has recently become a promising technique to overcome limitations with biological catalysts. Table 1 summarizes many of the reported examples that combine a number of different protein shells with various different catalysts. Among the methods employed are the direct fusion of a cargo enzyme with inward facing domains of the shell protein, the use of Coulombic attraction between shell and cargo protein domains, and the random (stochastic) capture of enzyme inside the particle during assembly. We have developed a system based on the capsid of the Qβ Levivirus, taking advantage of its well-known RNA packaging ability. In this system, packaging of a desired cargo protein is enhanced using a bifunctional RNA (biRNA) adapter, one part of which is designed to interact with the coat protein and the other with a peptide tag installed on the cargo. We have previously shown that this method can improve enzyme production, isolation, performance, and lifetime by sequestering enzyme catalysts in a protective biocompatible shell that allows small-molecule substrates and products to diffuse in and out.15 © XXXX American Chemical Society
Received: June 5, 2018 Revised: August 12, 2018
A
DOI: 10.1021/acs.biomac.8b00885 Biomacromolecules XXXX, XXX, XXX−XXX
B
a
58−64
58−64 58−64
56−65 42 × 72 42 × 72
E. coli
E. coli E. coli
E. coli Sf9 cells Sf9 cells, E. coli Sf 9 cells HEK-293 HEK-293
P22
P22 P22
HK97 Vault Vault
SV40 Lentivirus Retrovirus
15
16 17
18 19 20
21 22 23
GFP, yCD GFP, Linamarase GFP, FLP recombinase
GFP, mCherry GL, Luciferase GFP
CelB-GULK, GALK-GLUKCelB GFP, mCherry AdhD, CelB, NOX, Cas9
GFP, PhoA TFP EGFP, Luciferase IP1, SN, GFP, βgalactosidase, C99, GCSF, Cre EcHyd-1,CYP GalA, Hahead
DERA, yCD, PNP, SOD
HRP GFP, HIV protease GFP (+36), TOP(+36) RA, KE, βLac, CHAO, KatG, NOX,AldH SN EGFP, PalB Cytochrome P450 GFP, BFP, CFP, mCherry PepE, Luciferase
GFP
cargo protein(s)
vivo vitro vitro vivo vivo
vitro vivo vivo vitro vitro
vitro vivo vivo vivo
in vivo in vivo in vivo
in vivo in vivo in vitro
in vivo in vivo
in vivo
in vivo in vivo
in in in in
in vivo
in in in in in
in in in in in
in vitro
assembly conditionsa
225 aa gp4 protease tag 254 aa mINT domain tag 162 aa mINT domain plus Au particle plus hexa-His tag 50 aa interaction domain 200 aaVPR-PC tag N-terminal fusions to gag
141 aa scaffolding tag 141 aa scaffolding tag
141 aa scaffolding tag
141 aa scaffolding tag 141 aa scaffolding tag
FLAG or 16 aa Asp, Glu 29 aa C-term docking tag 37 aa C-termdocking tag 10 aa N-term capsid targeting sequence
bifunctional RNA/RNA
fusion to CP 21 aa coiled-coil domain cargo electrostatics bifunctional RNA bifunctional RNA
stochastic deca-Arg tag deca-Arg tag cargo electrostatics GFP(+36) fusion
stochastic
packaging mechanism or tag
2200 3000−5000
70 96 9
200−300 87−250
82, 15
109 183, 286
6.5, 3 12 ∼1−2 200
24, 36, 21, 18
240 15, 4 14 15 18, 10
1 4 14 100 45
95% if necessary. All purification steps after expression were performed at 4 °C to minimize protein degradation or catalytic inactivation. Characterization. The purity of assembled VLPs was assessed by isocratic size-exclusion chromatography with a Superose 6 column on an Akta Explorer FPLC instrument. Nonaggregated Qβ particles elute approximately 3 mL after the void-volume-associated peaks. TEM images were acquired with a Tecnai electron microscope (HP) with 100 kV, 1 s of exposure, and a charge-coupled device camera on carbon formavor grids stained with 2% uranyl acetate. The protein content of each sample was analyzed with a Bioanalyzer 2100 Protein 80 microfluidics chip. The average number of encapsidated proteins was determined by normalizing the area integration of coat protein and cargo protein peaks of purified VLPs to the calculated molecular weight of the proteins they signified, determining the molar ratio of coat protein to cargo protein and multiplying by 180 to obtain the number of cargo proteins loaded per VLP. This same analysis can be done with a sample of the bacterial culture after lysate, giving an estimate of the true ratios of expressed proteins that can be assembled into enzyme-packaged nanoparticles. Overall protein concentrations were determined with Coomassie Plus Protein Reagant (Pierce) according to the manufacturer’s instructions using bovine serum albumin for the standard concertation curve. We assume that the Coomassie Plus dye stains capsid and cargo protein to the same extent. Significance values were determined with a two-tailed Student’s t test. Representative characterization data of routine
the separate components be coexpressed in the Escherichia coli host to create multifunctional VLPs. This results in easy processing and scale-up but does not allow for precise control of assembly, producing an ensemble of particles with varying numbers of displayed and packaged domains. To modulate the average values of these parameters, indirect influencers of packaging need to be understood and controlled to consistently obtain the desired nanomaterial. We previously reported that environmental conditions such as the expression media, and expression temperature could influence packaging. Here, we investigate the effects on packaging of a test case (DERA) of the position of the Rev tag, the presence of the targeting bifunctional RNA, and expression from bi-cistronic mRNA.
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EXPERIMENTAL SECTION
Cloning. All sequences were verified by the direct sequencing of forward and reverse strands using unique primers at either ends (Genewiz). Plasmids were propagated in DH5a cells (BioPioneer) and grown in SOB (Amresco). The genes for the superoxide dismutase variants (sodA and sodC), 2-deoxyribose 5-phosphate aldolase (deoC), bacterial cytosine deaminase (codA), and purine nucleoside phosphorylase (deoD) were polymerase chain reaction (PCR)-amplified from the DH5α E. coli (BioPioneer, Inc.). The gene for uracil phosphoribotransferase (f ur1) was amplified from the genome of Saccharomyces cerevisiae (Champagne strain, Red Star) Primers used in this study are shown in Table S1. The FCY1 and tsFCY1 genes were codon-optimized in silico, and complete sequences were created by the gene assembly of overlapping DNA fragments (GeneDesign, Table S2). The 16 fragments were each diluted to 2 μM in a PCR mixture and cycled 55 times with an annealing temperature of 50 °C. This PCR reaction was purified by gel extraction, diluted 1:100, and used as the template for a second PCR using primers no. 13 and 14. To create pCDF−Rev-enzyme vectors, the PCR-amplified genes were digested with NcoI and XhoI, gel-purified, and ligated into a similarly digested pCDF vector coding for the synthetic Rev-peptide in-frame and directly upstream from the NcoI site. To create Histagged versions of the enzymes, PCR reactions using primer nos. 15 and 16 were used to amplify any Rev-enzyme construct. These were purified with gel extraction, digested with Kpn1 and Xho1, and ligated into similarly digested pCDF-1b. This created His-tag enzyme fusions with an eight-residue linker (GGASESGG). Untagged DERA was amplified with primer nos. 5 and 6, digested with NcoI and XhoI, and ligated into similarly digested pCDF-1b vector. To create C-terminal Rev-tag fusions, we first amplified the Rev-tag with an N-terminal linker and added a stop codon to the C-terminus with primer nos. 17 and 18. This PCR product was subcloned into pCDF-1b with BamHI and Xho1 to create pCDF-DERA-Rev. The gene sequence for the DERA was PCR-amplified with the enzyme forward primer nos. 5 and 19. This PCR product was then subcloned into pCDF-X-Rev with NcoI and BamHI to create pCDF-DERA-Rev. To create the dual Revtagged vectors, the DERA-Rev fusion was PCR-amplified with primer nos. 5 and 16. This product was subcloned into pCDF-Rev-enzyme with Nco1 and Xho1 to create pCDF Rev-DERA-Rev. The fusion between tsFCY1 and FUR1 was created by first PCR-amplifying the tsFCY1 with primer nos. 13 and 20 and ligated into any pCDF-Rev-X plasmid created above. This created the intermediate plasmid pCDFRev-tsFCY1-xsc. The f ur1 gene was amplified with primer nos. 12 and 21, digested with BamHI and Xho1, purified with gel extraction, and ligated into the similarly digested pCDF-Rev-tsFCY1-xsc. This created a genetic fusion of tsFCY1 and FUR1 with a three-residue (GSA) linker. Bi-cistronic vectors were created by first PCR-amplifying the Qβ coat protein (CP) gene with primer nos. 22 and 23, purifying with gel extraction, digesting with Nco1 and BamHI, and ligating into similarly cut pCDF-1b to create pCDF2-CP. A second PCR was then used to amplify anything within pCDF (enzyme, Rev-enzyme, enzyme-Rev, or C
DOI: 10.1021/acs.biomac.8b00885 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules particles are shown in Figure S1 and Table S3; data for the morespecialized particles both displaying and packaging functional polypeptides are shown in Figure 3. Free-Enzyme Production and Purification. The conditions used for expression of free enzymes were the same as used for the VLPs. To isolate the desired material, the cells were lysed in the presence of EDTA-free protease inhibitors (NEB), and the cleared cell lysate was passed through a cobalt−NTA Talon resin column (0.5 mL bed volume). The column was washed with 3 column volumes of PBS buffer, 3 volumes of PBS plus 20 mM imidazole, 2 volumes of PBS plus 100 mM imidazole, and eluted with PBS plus 300 mM imadazole. Fractions containing free enzyme were pooled and dialyzed against three changes of 1 L of PBS and concentrated with an Amicon Ultra centrifugal filtration unit (10 kDa MWCO, Millipore). All purification steps after expression and were performed at 4 °C to minimize protein degradation or catalytic inactivation. Purity was assayed by chip-based electrophoresis as above. Enzyme Activities. All enzyme activity experiments were run in triplicate at 25 °C unless noted and all replicates with encapsidated enzyme were performed in parallel with purified free enzyme for comparison. All assays were performed with respect to the overall enzyme concentration. Superoxide dismutase (SOD) activity was assayed with the SOD Assay Kit-WST (Dojindo) according to the manufacturer’s instructions. A total of 50% inhibition was considered at 1 unit per milliliter, and this was converted to units per milligram of enzyme for comparison among the various packaged and commercial versions. DERA activity was monitored with a coupled enzymatic assay described previously.60 In brief, approximately 50 nM DERA mediates the catalysis of 1 mM deoxyribose-5-phosphate (DR5P) to glyceraldehyde-3-phosphate and acetaldehyde. Acetaldehyde was reduced to ethanol by excess alcohol dehydrogenase (8 U/rxn) and reduced nicotinamide adenine dinucleotide (NADH, 0.3 mM) in Tris-HCl buffer (pH 8.5). We observed the conversion of NADH to NAD+ by monitoring the disappearance of absorbance at 340 nm in UV clear half-area well plates. From the molar absorptivity of NADH (6.22 mM−1 cm−1) and the amount of enzyme in each reaction, we calculated the specific activity (μmol/min/mg enzyme). For kinetic value measurement, we performed this reaction with 0−10 mM of DR5P and calculated the initial activity (nM of NADH/sec/nM of enzyme, sec−1) at each DR5P concentration and plotted these values by the DR5P concentration and fit this to the Michaelis−Menten equation. Cytosine deaminase activity and kinetics were analyzed by monitoring the UV absorbance at 234 nm of 5-fluorocytosine (5FC) using a Thermo Varioskan Flash plate reader with UV clear halfarea plates (Greiner-Bio). Molar absorbtivities (mM−1 cm−1) at 234 nm were determined with standard curves (5-FC, 6.62; 5-FU, 2.35). For determination of kinetic parameters, 40 μL of a 2x enzyme solution of His6-tsFCY1 or Qβ@(Rev-tsFCY1) was added to 40 μL of 0−1 mM substrate in PBS buffer and read immediately. A Michaelis− Menten nonlinear fit was used to obtain KM and kcat values. A total of 80 μL of solution volume in half-area well plates has a path length of 0.5 cm. Purine nucleoside phosphorylase (PNP) activity and kinetics were assayed by measuring the intensity of absorbance at 360 nm of the liberated 1-phosphate and 2-amino-6-mercapto-7-methyl- purine product from 2-amino-6-mercapto-7-methylpurine riboside (MESG) in the plate reader. Kinetic values were identified using a range of concentrations of the substrate (0−0.7 mM) in 50 mM Tris-HCl buffer (pH 7.5) with 1 mM PO4, 150 nM enzyme (final concentrations). Activity was initiated by injecting 40 μL of a 2× enzyme solution into 40 μL of substrate solution. The extinction coefficient of product at 360 nm was 1.93 mM−1 cm−1 and was used to convert absorbance to concentration. The initial velocities were plotted versus the concentration of the substrate, and a Michaelis− Menten nonlinear fit was used to obtain KM and kcat values. For protease protection studies with DERA, 140 nM of modified trypsin (NEB) was incubated with 200 nM DERA or Qβ@(DERA) and incubated at 25 °C. At the time points indicated, 8 μL aliquots were taken, and 72 μL of all of the reagents needed for activity
measurements were added and the initial rates measured. All data points were normalized to control treatments in which trypsin was not added. WT Qβ VLPs were added to the DERA samples to make the total protein concentrations in both samples equal. The assay that describes the long-term protection of packaged DERA compared to free DERA were the control samples that did not have protease in them. For protease protection studies with tsFCY1, 60 mU of trypsin (NEB) and chymotrypsin (MP Biomedicals) were added to 250 μL of buffer with 10× 6 μM His6-tsFCY1 or 12 μM Qβ@(Rev-tsFCY1)8 (enzyme concentration in PBS) and incubated at 25 °C. At the time points indicated, 8 μL aliquots were taken, and 72 μL of substrate (1 mM final concentration 5-FC) was added and initial rates measured. All data points were normalized to control treatments in which protease was not added. WT Qβ VLPs were added to the His6tsFCY1 samples to make the total protein concentrations in both samples equal. For thermal protection studies of DERA, 50 μL of a 200 nM solution (DERA concentration, in TBS) was incubated at the indicated temperature for 1 h. The solutions were then allowed to equilibrate to room temperature for 30 min before 40 μL was added to 40 μL of the 2× activity assay mix. Initial velocities for every incubation temperature were normalized to the initial velocity of the free or packaged DERA incubated at 4 °C. To determine the thermal half-life of cytosine deaminase and PNP enzymes, 60 μL of a 0.2 μM 2× solution (enzyme concentration in PBS) was incubated at 37 °C for a range of incubation times up to 48 h. Initial velocities for every incubation time were normalized to the initial velocity of the enzyme with no preincubation at 37 °C. Activity measurements were plotted vs time, and an exponential decay nonlinear fit was used to obtain half-life values. Cell Culture. All cell-culture reagents were purchased from Invitrogen unless noted otherwise. A431 cells were a gift from Carlos Barbas III (Scripps, La Jolla). Cells were grown and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% newborn calf serum (NCS; Omega Scientific), sodium pyruvate (1 mM), penicillin (100 units per milliliter), streptomycin (100 μg mL−1), and GlutaMAX (2 mM). HT-29 cells were grown and maintained in Roswell Park Memorial Institute (RPMI) medium supplemented as above. Cells were grown at 37 °C under humidified air with 5% CO2. Cells used for in vitro experiments were used at passage numbers less than 15. Cell-Viability Assays. HeLa cells were plated in 96-well microtiter plates in triplicate (5000 cells per well) in complete DMEM (RPMI for HT-29) (100 μL). After 18 h, the cytotoxic treatments (drug, prodrug) in serum-free medium (100 μL per well) at the indicated concentrations were added to the cells and allowed to incubate for 24 h at 37 °C unless otherwise noted. Treatments with VLP contained 4.2 μg of VLP, 30 nmole of enzyme, or both per well. The treatment media was removed and replaced with complete media. The cells were maintained at 37 °C with daily changes of media for 72 h. Cells were then assayed for viability by using a standard MTT assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg mL−1, 25 microliters per well) was added to each individual well, and the solutions were incubated at 37 °C for approximately 4 h (the assay was stopped when accumulated purple formazan crystals were visible in the control wells). The medium was carefully aspirated, and DMSO was added (200 μL per well) to dissolve the purple MTT−formazan crystals. Absorbance of the dissolved formazan was quantified at 570 nm by using a UV−vis plate reader, and cell viability was determined as a fraction of absorbance relative to untreated control wells. Data are presented as average values plus or minus standard deviation.
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RESULTS AND DISCUSSION
The Qβ packaging procedure involves the fusion of a desired cargo protein to the positively charged Rev peptide sequence at the N-terminus. Coexpression of this fusion with the Qβ bacteriophage coat protein (CP) and a bifunctional RNA D
DOI: 10.1021/acs.biomac.8b00885 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
forms of CD, 5−26 copies; and PNP, 16−21 copies. PNP represents the largest functional multimer to be packaged in the Qβ capsid. Yields of purified enzyme-containing particles were always greater than 20 milligrams per liter of culture and often greater than 50 mg/L, with a maximum value of 150 mg/ L in these studies. Determinants of Packaging. As described previously, expression media environmental conditions (composition, temperature, and time) can influence the number of cargo proteins that are packaged within the nanoparticle.15 In this report, the packaging number was quite variable for the enzymes tested, but clear environmental influences were not observed. In an attempt to reliably control the number of cargo proteins entrained during assembly, we used the DERA enzyme as the model cargo and focused on altering genetic parts of the packaging scheme including the position and number of the Rev-tags on the cargo protein, the presence of the bifunctional linker RNA, and the expression of the cargo and coat protein from the same (bicistronic) or different mRNA molecules. A complete discussion of this effort is given in the Supporting Information; the most important observations were as follows. Expression of the Cargo Protein: Moderate Inhibition by the Rev Tag and Position Importance. Significantly more untagged DERA was expressed relative to CP compared with all cases involving a Rev fusion (Table S4, entry 13); a similar phenomenon was observed earlier with packaged green fluorescent protein.27 Furthermore, more C-terminal Revtagged DERA was expressed relative to CP than N-terminal tagged enzyme (Table S4 and Figure 1a). Because it is unlikely
(biRNA) molecule noncovalently recruits Rev-tagged cargo to the assembling particle by virtue of aptamer sequences specific for CP and Rev in the transcribed but not translated RNA (Figure S1).15 Assembly of 180 CPs into the canonical T = 3 icosahedral Qβ particle occurs spontaneously in the E. coliexpression host, with biRNA and cargo protein inside. In this study, the procedure was extended to the new enzymes, with the products designated Qβ@(Enz)n, where n represents the average number of cargo proteins encapsidated per nanoparticle. This value was determined from the relative intensities of bands observed in microfluidic denaturing electrophoresis (Agilent Bioanalyzer P80 chip), normalized by their respective molecular weights. Note that “R-Enz” designates a fusion protein in which the Rev tag was incorporated at the Nterminus of the enzyme, “Enz-R” designated a C-terminal Rev tag, and “R-Enz-R” refers to a cargo enzyme having Rev tags at each end. Modular Packaging of Different Enzymes within the Nanoparticle. The previously described Qβ packaging scheme15 is amenable to many different types of cargo proteins. Simple cloning operations allow the rapid insertion of genes into the appropriate plasmid, and a standardized protocol of particle expression, isolation, and purification was used for every cargo tested. We report here the use of this modular method for the packaging of four different industrial and therapeutically relevant cargo enzymes. The following catalysts were chosen for their practical importance and familiarity as enzymological standards: 2deoxyribose 5-phosphate aldolase (DERA, gene deoC,45 a core metabolic enzyme with potential as an enantiospecific catalyst for the production of chiral compounds on industrial scale),61 superoxide dismutase A (SodA, a cytoplasmic homodimer that uses Mn ion in its active site),62 cytosine deaminase (CD, which hydrolyzes cytosine to uracil and also converts the fungicide 5-fluorocytosine to the toxic antimetabolic 5fluorouracil, thus functioning as a popular prodrug-converting enzyme),52,63 and E. coli purine-nucleoside phosphorylase (PNP, 26 kDa, active as a hexamer in vivo).55,56 A total of four variants of cytosine deaminase were packaged: codA from E. coli.,64 FCY1 from S. cerevisae, a thermostable triple mutant of this enzyme (tsFCY1),65 and a fusion between tsFCY1 and uracil phosphoribotransferase (FCU1).66,67 In each case, the codon-optimized gene coding for each enzyme was augmented with the sequence for an N-terminal Rev-tag and was expressed in E. coli with the Qβ capsid protein in the standard manner designed to induce encapsidation during VLP self-assembly. Because the properties of the VLP do not vary with the nature of the entrained protein, a single standard VLP isolation protocol was performed in all cases, ending with isolation from the appropriate band in a sucrose gradient after ultracentrifugation (see the Supporting Information). Occasionally, a single such gradient was not sufficient to remove all contaminants such as small (∼4 kDa) peptide fragments; in those cases, repeat sucrose gradient purification provided products in which CP and cargo proteins comprised greater than 95% of the total protein. All of the enzyme-containing particles were indistinguishable from wildtype particles (lacking packaged protein) by size-exclusion chromatography (SEC) and transmission electron microscopy (TEM); see the Supporting Information for representative data. The average numbers of packaged proteins varied from run to run, with the following ranges of the number of enzyme monomers: DERA, 5−18 copies; SodA, 9−18 copies; various
Figure 1. Box plots of the combined data from Table S4 highlighting significant differences. (a) Plots of the packaging number (no. in VLP or no. expressed) by the placement of the Rev tag. (b) Plots of the packaging efficiency (Peff) by the number of Rev tag(s). Significance values: single asterisks indicate p < 0.05, double asterisks indicate p < 0.01, and triple asterisks indicate p < 0.001.
that the position of the Rev-tag on DERA impacts expression of CP, this suggests that N-terminal Rev-tagging interferes with expression of R-DERA and R-DERA-R proteins. This could be due to interference with ribosome binding, translation initiation, or, perhaps, translation elongation. Packaging and Strong Enabling by the Rev Tag. Although expressed in large amounts relative to coat protein, untagged DERA was very poorly packaged in assembled particles (Table S4, entry 13). Packaging efficiency (Peff) is defined as the ratio of packaged to expressed cargo protein, each relative to coat protein (the vast majority of which is assembled into particles). As previously observed to varying degrees with peptidase-E E
DOI: 10.1021/acs.biomac.8b00885 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. Schematic of two different co-expression schemes to produce nanoparticles that package cargo protein and display targeting motifs. (a) Co-expression of bicistronic plasmid coding for the CP, CP-X, and biRNA and a separate plasmid coding for the Rev-tagged enzyme. (b) Coexpression of a plasmid coding for the CP-X protein with a separate bicistronic plasmid coding for the CP, Rev enzyme, and biRNA.
Table 2. Tests of Different Co-expression Schemes for Simultaneous Enzyme Packaging and Polypeptide Display entry
gene following CP in pET282HaR bi-cistronic (no. amino acids in extension)
gene in pCDF (no. amino acids in extension)
yielda
no. enzymes per VLP
no. ligands on exterior per VLP
1
R-tsFCY1
−
++
13−36
−
2
R-tsFCY1
CP-ZZ (68)
++
8−22
36−53
3
R-tsFCY1
CP-EGF (102)
+
6−23
2−5
4 5 6
R-tsFCY1 R-tsFCY1 R-PNP
CP-GE7 (12) CP-F56 (18) −
++ − ++
6 7, 14 16−21
50 10, 19 −
7 8 9 10
R-PNP R-PNP R-PNP CP-ZZ (68)
CP-ZZ (68) CP-EGF (102) CP-GE7 (12) −
++ + − +
10 15 10 −
29 5 13 31−41
11 12 13 14 15 17 18 19 20 21 22
CP-ZZ (68) CP-ZZ (68) CP-ZZ (68) CP-ZZ (68) CP-ZZ (68) CP-GE7 (12) CP-GE7 (12) CP-GE7 (12) R-tsFCY1 R-tsFCY1 R-PNP
R-tsFCY1 R-tsFCY1 R-PNP R-PNP R-SodA − − R-PNP R-PNP R-UPRT R-tsFCY1
++ + ++ ++ + + + ++ ++ +
5 10 11 16 11 − − 12 3,8 4 8
50 27 38 36 38 26, 36 54 47 4, 8b 4b 8b
expression media MEM, SOB MEM, SOB MEM, SOB SOB SOB MEM, SOB SOB SOB SOB MEM, SOB SOB MEM SOB SOB SOB SOB SOB SOB SOB SOB SOB
expression temp. (°C)
N
30
6
30
4
30
5
30 30 37
1 2 3
37 37 37 30, 37
1 1 1 3
30 30 30 37 37 37 30 37 30 30 30
1 1 1 1 1 2 1 1 2 1 1
a
VLP yields >20 mg/L of culture (++), 3−20 mg/L of culture (+), or 0.9), in which enzyme concentration is calculated on the assumption that all of the packaged proteins are equivalently active. Commercially available DERA enzyme proved to be much less active (specific activity: 2 μmol/min/mg enzyme) and so was not included in kinetic analyses. The kinetic parameters of the three DERA enzyme samples (Table 3) were measured and show interesting differences. Free DERA has a turnover number (kcat = 9.1 ± 0.2 s−1) midway between the values observed for doubly Rev-tagged I
DOI: 10.1021/acs.biomac.8b00885 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 6. Characterization of the protection the nanoparticle confers to DERA. (a) Relative activities of packaged and free DERA after incubation in the presence of 140 nM trypsin at the indicated time points 25 °C. Values are normalized to control samples to which protease was not added. (b) Relative activity of the packaged and free DERA after long-term storage at 25 °C. Values are normalized to the activity at t = 0. (c) Relative activities of packaged and free DERA after 1 h of heating at the indicated temperatures, followed by a return to 25 °C for measurement of enzymatic activity. (a, b) Displayed values are the average of triplicates plus or minus standard deviations.
be in the range between 8 and 12 h at 37 °C, compared to 5 h for free His-tagged tsFCY1 under the same conditions. The packaged protein was also largely resistant to protease treatment (trypsin and chymotrypsin) under conditions that completely inactivated free tsFCY1 (Figure 8a,b). The
shown to affect the kinetics of enzymes previously packaged within VLPs derived from Qβ,15 MS2,28 P22,33,36 and CCMV.25 To investigate if tsFCY1 was sensitive to lumenal concentration, we measured the kinetic constants (kcat and KM) of nanoparticles containing different amounts of the enzymes, as well as with displayed peptides. Figure 7 shows these kinetic
Figure 8. Functional characterization of packaged cytosine deaminase. (a, b) Incubation of packaged and free tsFCY1 in the presence of nonspecific proteases. (a) Incubation in the presence of trypsin. (b) Incubation in the presence of chymotrypsin. Data are presented as average of triplicates plus or minus the standard deviation and normalized to samples with no protease added. The lines are best-fit curves. Qβ@(R-tsFCY1) is shown by black squares and solid lines and His6-tsFCY1 is shown by red diamonds and dashed lines.
anticipated production of cytotoxic 5-FU was validated by measurement of the ability of the prodrug−enzyme combination to kill mammalian cells in vitro (Figure S4). In the presence of Qβ@(Rev-tsFCY1)21, 5-FC exhibited an EC50 value (5 μM) equivalent within experimental error to that of 5FU administered alone (17 μM). These values were similar to those obtained in the context of antibody-directed stem cell elimination in vitro.76 Purine Nucleoside Phosphorylase. Nanoparticles containing PNP were fairly active (kcat = 4 s−1) toward the fluorescent substrate MESG77 and displayed a half-life at 37 °C of 12 h, long enough to be of potential use for in vivo applications. However, the apparent therapeutic window was poor: in our hands, prodrug fludarabine was only marginally less toxic than the released F-Ade, whereas alternative prodrug 2-fluoroadenosine (F-Ado) was actually more toxic than F-Ade.
Figure 7. Kinetic constants of the all the Qβ@(R-tsFCY1) nanoparticles generated in this study. (a) Michaelis constant (KM) vs the number of packaged enzymes per VLP or [E]conf. (b) Plot of the turnover number (kcat) vs the number of enzymes per VLP. (c) Plot of kcat vs KM.
parameters as a function of the internal density. Within some subsets, such as ZZ- and EGF-displaying particles, the kinetic parameters were quite reproducible and insensitive to the number of packaged enzymes. Other series showed variations in kinetic parameters of almost a factor of 5, but no clear trends emerged other than perhaps a weak compensatory correlation between kcat and KM (Figure 7c). Nonetheless, all of the packaged enzymes showed greater activity than the free enzymes at the therapeutic relevant dosage of 150 μM of 5-FC. The half-lives of the packaged enzymes at 37 °C were found to
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CONCLUSIONS RNA-directed enzyme packaging in Qβ VLPs has been shown here to depend more on charge complementarity to the appended Rev tag than on a specific interaction of the tag with J
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a cognate aptamer sequence. The system is quite robust, however, being successful in packaging a variety of enzymes of different functional aggregation state in forms allowing for highly catalytic activities. Some additional elements of control over packaging were identified. For example, N-terminal Rev tagging proved to be detrimental to cargo protein expression and to nanoparticle encapsulation, whereas Rev tagging at both the C- and N-termini greatly enhanced packaging efficiency. Because the VLP shell is easy to isolate and purify and is resistant to protease digestion and thermal denaturing, this system for production of packaged proteins is quite processfriendly. The entrained enzymes are often more amenable to storage at room temperature or for long periods than the free enyzmes and are effectively protected from protease attack. The enzymes tested here do not appear to be affected by packaging density, but the heterogeneity of enzyme activity values may obscure subtle trends. We also show here for the first time a convenient and modular technique for encoding both the external display of functional polypeptides and the internal encapsidation of functional enzymes in the same particles.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00885. Primer and gene synthesis sequences, representative particle characterization data, and descriptions of additional experiments used to optimize the packaging system. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mgfi
[email protected]. ORCID
M. G. Finn: 0000-0001-8247-3108 Funding
This work was supported by the National Institutes of Health (grant nos. GM101421 and EB015663) and the Skaggs Institute for Chemical Biology. Notes
The authors declare no competing financial interest.
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