Alison
O'Neil
a,
Peter E.
Prevelige
b and
Trevor
Douglas
*a
aDepartment of Chemistry and Biochemistry, Center for BioInspired Nanomaterials, Montana State University, Bozeman, MT 59717, USA. E-mail: tdouglas@chemistry.montana.edu; Fax: +1 406-994-5407; Tel: +1 406-994-6566
bDepartment of Microbiology, University of Alabama, Birmingham, AL 35294, USA
First published on 17th June 2013
Virus like particles, and other naturally occurring protein containers, have emerged as excellent building blocks for nanomaterials design and synthesis. Here we exploit a directed assembly and encapsulation approach to sequester multiple copies of a phosphotriesterase (PTE) enzyme within the capsid of bacteriophage P22. Phosphotriesterase, from Brevundimonas diminuta, is an intriguing enzyme as it is highly active against a wide range of harmful insecticides and nerve agents such as Soman and Sarin. However, difficulty in expressing large quantities of the active recombinant enzyme has limited efforts to scale-up its use. Additionally, as a mesophilic enzyme its low heat tolerance and susceptibility to proteolysis makes it a less than ideal candidate as a practical bioremediation tool. Through encapsulation of the PTE within the P22 capsid, we demonstrate a greatly enhanced thermal tolerance of the enzyme, maintaining 50% of its activity to 60 °C. Additionally, the P22 capsid confers protection to the enzyme from proteases, as well as stabilizing the enzyme against desiccation. Thus, our engineered P22 encapsulation system greatly enhances the stability of the mesophilic phosphotriesterase and results in a robust and active nanoparticle reactor.
Upon encapsulation of enzymes in protein containers we, and others, have observed varied effects on the enzyme kinetics. In some cases, there is little to no effect,5,11 in others the overall rate seemed to be enhanced upon encapsulation,9,12 while in other examples the rate (kcat) and the KM were decreased.5 The kinetic trends and outcomes of high density encapsulation is not yet predictable for this promising new class of biomaterials and at present appear to be enzyme specific.
Viral capsids are very robust and can be utilized to protect non-native cargo, analogous to their natural nucleic acid protection, in a biomimetic approach to developing new active nanomaterials. Many virus like particles (VLPs) assemble into precise architectures, can be stable for long periods of time, and some can withstand dramatic pH changes.13 In the case of the bacteriophage P22 capsid, the VLP is soluble to high concentrations (30 mg mL−1), highly resistant to proteases, maintains the capsid assembly across a wide pH (pH 4–10) and temperature (−80 to 85 °C) range, can be lyophilized and rehydrated, and can withstand high concentrations of organics all without disrupting the cage like architecture of the VLP capsid. By exploiting this capsid as a nanocontainer for directed encapsulation of an enzymatic cargo, we hoped to bestow these stabilizing qualities on the cargo enzyme and use this advantageously towards the engineering of novel active nanocatalyst materials.
We have investigated the encapsulation of the highly active phosphotriesterase (PTE) from Brevundimonas diminuta, which has been shown to break down organophosphates, including chemical warfare agents and commercial insecticides.14 The homodimeric, mesophilic enzyme has been shown to contain a bi-nuclear zinc active site, which can be replaced with cobalt to generate an even more active enzyme.15,16 The high catalytic activity, and toxic substrate remediation, makes PTE an intriguing candidate for large-scale bioremediation. However, large-scale isolation of the enzyme for practical applications has been hindered due to the difficulty in scale-up expression of active enzyme.17 When over-expressed in E. coli, the recombinant PTE was found largely in an aggregated inactive form and active enzyme made up only ∼0.1% of the total cell mass.17 Additionally, as a mesophilic enzyme, PTE has low heat tolerance (loses activity above 45 °C) when free in solution or immobilized on a resin.18 Therefore, as promising a bioremediation tool as the PTE may be, the poor expression yields and low heat tolerance makes it less applicable for ultimate industrial use. These properties make PTE a prime candidate for protein expression rescue and thermal stability enhancement via incorporation into our synthetic bio-materials approach. Through the encapsulation of PTE within the P22 capsid, we expand the usefulness of this enzyme by increasing the active protein yield in E. coli and by greatly increasing the thermal tolerance of PTE. Our engineered system confers additional advantages by protecting the mesophilic enzymes from proteolytic degradation and lyophilization.
The ligated plasmid was transformed into XL-2Blue ultra competent cells (Agilent Technologies, Santa Clara, CA) and colonies were screened via single pass sequencing for the incorporation of the three genes. The confirmed sequence fusion plasmids were subsequently transformed into BL21 E. coli (EMD Chemicals) for protein expression.
For the P22 encapsulated samples, clarified lysate (25 mL) was layered on top of a 35% (w/v) sucrose cushion (5 mL) and centrifuged at 48000 rpm for 50 minutes in an ultra centrifuge (Sorvall WX Ultra). The resulting virus pellet was resuspended in 50 mmol L−1 HEPES buffer pH 8.5 and spun at 17000g for 20 minutes to remove any particulates and lipid. The virus was then dialyzed against the aforementioned buffer overnight.
The unencapsulated free phosphotriesterase fusion was purified out of the ultra centrifugation supernatant produced in the step above. The clarified supernatant was loaded onto a HisTrap HP Column (Amersham) using an AKTA FPLC (GE Healthcare). Samples were washed with imidazole (20 mmol L−1) in phosphate (20 mmol L−1, 500 mmol L−1 NaCl, pH 8). Protein was eluted with imidazole (250 mmol L−1) and dialyzed against HEPES buffer (50 mmol L−1, pH 8.5) extensively.
Fig. 1 Through the P22 encapsulation system, phosphotriesterase (PTE) is specifically sequestered on the interior of the P22 capsid and maintains its catalytic activity. (a) Cartoon representation illustrating the plasmid design on the left and the spontaneously assembled product on the right. (b) Negatively stained TEM image of the P22-PTE capsids. Bar represents 100 nm. (c) As the P22-PTE cleaves the paraoxon substrate, a p-nitrophenol group is released which can be monitored as an increase in the absorbance at 405 nm. |
To further understand the role that the scaffold protein expression plays in the capsid assembly, a construct expressing only the P22 coat protein was made. Identical expression conditions were used and the resulting “coat protein only” materials were subjected to size-exclusion chromatography to assess the relative amount of properly assembled capsids. The SEC profile revealed a significant peak that eluted early and corresponds to a population that is mis-assembled into a wide range of irregular structures. A slightly later eluting peak corresponds to well-formed (T = 7) P22 capsid particles. This profile is similar to what was observed with the P22-PTE construct grown under non-optimized conditions. In both cases the ratio of the early (mis-assembled/aggregate) peak to the later eluting (properly assembled) peak, was much higher than what is seen in constructs where the scaffold fusions are well expressed and present in high copy number during the assembly process (Fig. S3†). With the poorly expressing (or no) scaffold protein constructs, the mis-assembled and assembled peaks are not perfectly resolved by SEC, indicating some heterogeneity in the material. When examined by TEM, the images of the poorly expressing (or no scaffold protein) constructs revealed many multiple shelled and irregular capsids (Fig. S4†). These results indicate that the scaffold protein fusion expression level is central to the proper templating of our P22 nanomaterial assembly.
However, despite these challenges, after the optimization of growth conditions and size-exclusion purification, the P22 encapsulation system still produces homogeneous material with good yields (∼150 mg of purified material per liter of E. coli grown or ∼5 g cell paste). Analysis of the purified P22-PTE material by HPLC-SEC coupled to quasi-elastic and multi-angle light scattering (MALS) detectors, revealed that the purified capsids were homogeneous in size with a radius of hydration (RH) of 30.57 ± 0.12 nm and a radius of gyration (Rg) of 27.87 ± 0.06 nm. The ratio of Rg/RH for empty P22 is 0.95, which is characteristic of an empty sphere, where as with P22-PTE this ratio is 0.91 suggesting packaging of the cargo enzyme. From MALS, the average total molecular weight for the P22-PTE capsids was 21.8 ± 0.56 MDa as compared to 19.65 MDa for empty P22, indicating packaging of 40 ± 10 copies of the PTE-SP fusions per capsid (see ESI† for calculation).
The enzymatic activity of the encapsulated PTE was monitored at room temperature across 11 substrate concentrations ranging from 6.7 μmol L−1 to 1.2 mmol L−1. Paraoxon was used as the substrate because its enzymatic degradation product p-nitrophenol gives a characteristic absorbance at 405 nm. For each substrate concentration, the rate of paraoxon hydrolysis was determined based on the change in absorbance at 405 nm over 5 minutes (Fig. 1c). Fitting this data to a Michaelis–Menten model gave a KM of 136 ± 15 μmol L−1 and a kcat of 10.7 ± 0.5 s−1 (Fig. 2). The reported values of KM and kcat, for the unencapsulated cobalt substituted enzyme are 100 μmol and 4870 ± 90 s−1 respectively.14 Encapsulation of enzymes in protein containers has been observed to produce varied effects on the kinetics of the enzyme. In the case of P22-PTE, the enzymes KM is slightly increased and the turnover (kcat) severely decreased. This observation may indicate that not all of the encapsulated enzymes are actually functional or are distorted such that they do not function as well as the PTE-fusion free in solution. However, like classical enzymology, characterizing the individual number of active proteins within a purified preparation is difficult. Considering the P22-PTE as a multi-component nanoreactor, with 40 active enzymes per capsid, the per capsid turnover rate is 400 s−1, still an excellent active nanoparticle catalyst.
Fig. 2 P22-PTE kinetics were measured using paraoxon as a substrate between 6.7 μM–1.2 mM. The plotted rates were fit to a Michaelis–Menten model (R2 = 0.99) to determine a Vmax of 0.096 ± 0.003 and a KM of 136 ± 15 μM. |
Fig. 3 Encapsulation of phosphotriesterase within the P22 capsid greatly decreases its thermal sensitivity. Samples were heated at the indicated temperatures for 20 minutes, cooled to room temperature and assayed for activity. While the unencapsulated enzyme quickly loses activity with heat, the encapsulated enzyme maintains >70% activity to 55 °C. At 60 °C, the unencapsulated enzyme has lost almost all activity while the encapsulated maintains ∼45% activity. |
The P22 capsid confers an additional level of protection to the encapsulated PTE in that it shields the enzyme from access to proteolytic digestion. P22-PTE and the unencapsulated PTE were treated with an excess of the protease trypsin for either one hour or over-night at room temperature and then assayed for activity. After one hour trypsin treatment, the unencapsulated PTE lost 67% of its activity while the activity of the encapsulated PTE remained unchanged (Fig. 4). Overnight (18 hours) trypsin treatment reduced the unencapsulated PTE activity to 19% of the untreated while the P22 encapsulated PTE maintained greater than 90% of its original activity (Fig. 4). This enhancement of the enzyme stability makes the P22-PTE a significantly better candidate for remediation applications. If PTE were used to remediate insecticide-contaminated wastewater, the enzyme would likely encounter many proteases from an environmental sample. By protecting the enzyme inside the P22 capsid, which is porous enough for small substrate molecules, but restrictive of larger proteases, PTE is better suited for practical applications.
Fig. 4 Encapsulation of the phosphotriesterase (PTE) within the P22 capsid confers heightened resistance to proteolytic degradation and loss of activity after rehydration. Treatment with trypsin for either one hour or overnight greatly decreased the activity of the unencapsulated PTE while the encapsulated PTE maintained activity with either length of treatment. Upon lyophilization and subsequent rehydration, the unencapsulated enzyme lost all activity while the encapsulated enzyme retained 91% of its activity. |
Additionally, when the P22-PTE and the free enzyme were lyophilized and then rehydrated, the encapsulated enzyme retained 91% activity while the free enzyme lost all activity (Fig. 4). For long-term storage and use, it may be advantageous to be able to freeze dry active P22-PTE material. Through encapsulation within the P22 capsid, this storage method is now compatible with the PTE enzyme. It is also noteworthy that during the several months of this study, the P22-PTE stored in solution at 4 °C did not lose any measurable activity. However the free-PTE was unstable in solution and slowly precipitated under the same conditions and exhibited inconsistent enzyme activity. The encapsulation of PTE in the P22 capsid thus confers many desirable materials properties to the enzyme making it a more advantageous candidate for incorporation into larger scale materials synthesis.
The authors would like to thank Professor Frank M. Raushel (Texas A and M University) for supplying the phosphotriesterase gene. Ms O'Neil held an AAUW American Fellowship while conducting this research. This research was supported by a grant from the National Science Foundation (BMAT-1104876).
Footnote |
† Electronic supplementary information (ESI) available: Calculations, SEC chromatograms, and TEM micrographs. See DOI: 10.1039/c3bm60063g |
This journal is © The Royal Society of Chemistry 2013 |