- Research article
- Open Access
Identification of fragments from Autographa Californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation
© Sampieri et al.; licensee BioMed Central. 2015
- Received: 16 July 2014
- Accepted: 15 January 2015
- Published: 4 February 2015
Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied.
Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1–110 contains two repeats formed each of two β sheets followed by a α helix (amino acids 1–58 and 58–110) that are important for the formation and stability of polyhedra. These fragments 1–58, 58–110 and 1–110 could be incorporated into polyhedra. However, only fragments 1–110 and 58–110 can self-aggregate.
These results demonstrate that 58–110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra.
- Autographa Californica
The development of eukaryotic systems for the expression of recombinant proteins has been a major goal in biotechnology due to its widespread utility in medicine, veterinary medicine, and agriculture, among other related areas .
The use of insect viruses to produce and to obtain different recombinant proteins has grown in recent decades [2,3]. Three of these eukaryotic systems are expressed in insect cells and are currently in use. Two of them are based on the DNA baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV), and Bombyx mori nucleopolyhedrovirus (BmNPV). The third uses the RNA virus Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) cypovirus. In nature, the viruses of these 2 families are protected from adverse environmental conditions as they are occluded into crystalline lattices called polyhedra or occlusion bodies, derived mainly from a single viral protein called polyhedrin . The occlusion is an adaptation that allows baculoviruses to remain in a dormant but infective state in the environment for decades .
Polyhedrin is one of the most abundant proteins in a baculovirus-infected cell, since its expression is driven by a very strong promoter . Because polyhedrin is not necessary for the propagation of the virus, the DNA sequence of the protein can be replaced with some other sequence of interest . This in turn, has allowed the polyhedrin promoter to be used as an expression strategy for obtaining high yields of recombinant proteins.
Since BmNPV and BmCPV polyhedra are particles of about 1 μM in diameter and can be easily purified by centrifugation, they represent good candidates to express recombinant proteins. Using this strategy, Je et al. incorporated the green fluorescent protein (GFP) into the AcMNPV polyhedra by fusing it to the carboxyl terminus from the polyhedrin gene . However, the expression of the recombinant protein did not form polyhedra . Only the combined expression of both the wild type (WT) and the recombinant polyhedrin (GFP-polyhedrin) proteins resulted in the formation of polyhedra . This result shows that fusing proteins to polyhedrin prevent the formation of polyhedra, but WT polyhedrin can rescue this phenotype. Nevertheless, these results highlight the little we understand about how polyhedra particles are formed in the nucleus of baculovirus infected cells.
The polyhedrins of baculoviruses and cypoviruses do not share a similar amino acid sequence . However, the crystal structures of both polyhedra are indistinguishable between the two families in terms of their size and symmetry [10,11]. Thus, these conserved properties suggest that the crystal structure of polyhedra has been retained in nature for the specific purpose of preserving the viruses, and that such crystalline structure can be obtained using proteins with different amino acids compositions.
It has already been shown that the crystal structure from both the AcMNPV and the BmCPV polyhedra is an arrangement of polyhedrin trimers, which are interconnected through their amino N-terminal helices . These interactions provide significant stability to the polyhedra, since the trimer is the base of the crystal core . The identification of the properties of the crystallography structure has allowed investigators to determine the interacting amino acids in the crystal formation and to identify which of them are necessary for configuring the polyhedra core structure [11,10].
Despite the similarities in crystal symmetries and identical unit cell dimensions, structures of baculovirus and cypovirus polyhedrins are different at the atomic level. Both structures have a β-sandwich core domain, with projecting C- and N-terminal helices, but the topologies are dissimilar and the helices interact differently .
Based on these findings, Ijiri et al. incorporated several foreign proteins into BmCPV polyhedra by fusing them to the first 30 amino acids of polyhedrin, which contains an α helix known as H1 . Because this fragment projects towards the outside of the protein, it forms independently as the molecule folds; it interacts with other molecules of polyhedrin and it is incorporated into polyhedra crystal structure. Thus, the co-expression of H1 with the WT polyhedrin is now widely used as a tag to incorporate foreign proteins into BmCPV polyhedra [13-15].
More recently, recombinant polyhedra in BmNPV have been obtained by co-expressing the foreign proteins fused to the first 110 amino acid N-terminal fragment in combination with the complete WT polyhedrin [16,17]. The foreign proteins were then purified from polyhedra [16,17].
These findings unravel the role of the amino region 58–110 from polyhedrin in the assembly of AcMNPV polyhedra, while providing the bases for a system to incorporate efficiently high levels of recombinant proteins into the polyhedra crystal.
Insect cell line and baculoviruses
The Spodoptera frugiperda cell line, Sf9, was maintained at 27°C in Grace medium (Invitrogen, USA) supplemented with 10% heat-inactivated fetal bovine serum (56°C, 30 min) (Wisent, Inc., Canada), 1X Yeastolate (Invitrogen, USA), 1X Lactalbumin (SIGMA, USA), and 1X Antibiotic-antimycotic (Invitrogen, USA) according to standard methods. For suspension cultures, pluronic acid F-68 at a final concentration of 0.1% was added and the cells were sub-cultured every 2 to 3 days. The Bac-N-blue system (Invitrogen, USA) was used for the construction of the recombinant baculovirus (see below). The WT and recombinant AcMNPVs used in the present study were propagated in Sf9 cells.
Generation of the recombinant baculovirus
The complete polyhedrin gene was obtained by PCR amplification using the Baculovirus forward and reverse PCR primers (Invitrogen, USA) and cloned into pEGFP-C2 (Clontech, USA). The recombinant baculoviruses with the different fragments of polyhedrin were constructed by digestion with restriction enzymes or by PCR amplification of the constructions. These PCR fragments were cloned to pEGFP-N1, −N2 or -N3 (Clontech, USA) as needed to obtain the fusion proteins with an open reading frame. The recombinant plasmid vectors were confirmed by restriction endonuclease analysis and sequencing. All the fusion genes of EGFP-polyhedrin were then sub-cloned into the pBlueBac4 plasmid (Invitrogen, USA), which was used with the Bac-N-blue transfection kit (Invitrogen, USA) to obtain the recombinant baculoviruses. The recombinant baculoviruses were purified and then amplified to obtain high titer virus stocks. The baculovirus titer was obtained and expressed as plaque forming units (pfu) per milliliter according to standard protocols provided by the manufacturer (Invitrogen, USA).
Production and purification of recombinant polyhedra
Sf9 cells co-infected with the recombinant and WT viruses were collected by centrifugation at 96 hrs post-infection, resuspended in phosphate buffered saline (PBS; 20 mmol/L NaH2PO4, 20 nmol/L Na2HPO4, 150 mmol/L NaCl, pH 7.2) (Sigma, USA), and fragmented ultrasonically three times for 30 s each (Braun Biotech International, Germany), followed by centrifugation at 12,000 g at 4°C for 10 min. The pellets were then washed 2X with PBS and then finally resuspended in PBS buffer.
Confocal microscopy 3D reconstructions and electron microscopy scanning
The recombinant purified polyhedra of infected insect cells were allowed to adhere to each of the wells of a LabTek II Chamber Slide (NalgeNunc Int, USA). The polyhedra or cells were washed three times with PBS and fixed with mounting medium (DakoCytomation, USA). The infected insect cells were incubated with DAPI (4,6-Diamidino-2-Phenylindole, Dihydrochloride), Molecular Probes, USA) at a dilution 1:1000 for 5 min before fixation with DAKO Cytomation fluorescence mounting media (DakoCytomation, Denmark). Images were collected with an Olympus FV1000 confocal microscope and analyzed using Fluoview 10-ASW-2.1 software (Olympus, Japan). Image acquisition using transmission electron microscopy (TEM) of infected cells was conducted according to established protocols. Briefly, the cells were washed with 0.08 M cacodylate buffer (Sigma St. Louis, MO) and fixed for 10 min with 0.6% glutaraldehyde (Sigma St. Louis, MO) and 0.4% paraformaldehyde in 0.08 M cacodylate buffer, pH 7.4. Post-fixation was made with 1% osmium tetroxide (Fluka, St. Louis, MO) in cacodylate buffer Thin sections were counterstained with uranyl acetate for 10 minutes and with lead citrate for 2.5 minutes. Observations were made in a Jeol 1010 electron microscope (Jeol USA, Peabody, MA).
Flow cytometry studies
Nanoparticle Tracking Analysis (NTA)
The crystal structure of the wild type AcMNPV polyhedrin (2WUY.pdb, http://www.rcsb.org/pdb/explore.do?structureId=2wuy)) was utilized in the identification of the polyhedrin secondary structure, which directed the generation of the different polyhedrin fragments reported in this study.
Recombinant baculoviruses expressing polyhedrin fused to EGFP
In order to visualize the expression and localization of polyhedrin and polyhedra in the infected cells, we developed several recombinant baculoviruses containing different fragments from the polyhedrin gene fused to the enhanced green fluorescent protein (EGFP). The baculovirus were designated according to the amino acid fragment of polyhedrin: WT polyhedrin (PH(1–245)-EGFP), and the different fragments of polyhedrin: amino acids 1–25 (PH(1–25)EGFP), 1–34 (PH(1–34)EGFP), 1–48 (PH(1–48)EGFP), 1–58 (PH(1–58)EGFP), 17–58 (PH(17–58)EGFP), 58–110 (PH(58–110)EGFP), 1–110 (PH(1–110)EGFP), and 111–245 (PH(111–245)EGFP). All recombinant genes were under the control of the polyhedrin promoter in the recombinant baculoviruses. All polyhedrin fragments were cloned at the N-terminal of the EGFP except for PH(1–245)EGFP which was cloned at both the N- and C-terminal of the EGFP. Because we obtained indistinguishable results with both constructs, we will describe here only PH(1–245)-EGFP. In order to obtain polyhedra, a second recombinant baculovirus carrying the wild type copy of polyhedrin was utilized in co-infections with all baculoviruses carrying fragments from the polyhedrin gene (listed above).
Recombinant PH(1–245)-EGFP was expressed in the cytoplasm of Sf9 infected cells but it retains the ability to form aggregates
WT polyhedrin is required for the formation of polyhedra
Polyhedra-like formation in Sf9 cells infected with PH(1–110)EGFP baculovirus
TEM images from cells expressing PH(1–110)EGFP showed electron dense intranuclear protein aggregates, corresponding to the polyhedralike structures observed in confocal microscopy (Figure 6C). Notice that these aggregates did not contained baculoviruses; rather viruses appeared to be discarded from the aggregates (insets C’ and C”). Similar results were obtained with the full length PH(1–245)-EGFP recombinant polyhedrin (data not shown).
Co-expression of PH(1–110)EGFP with WT polyhedrin resulted in the formation of canonical polyhedra with EGFP contained within the crystal (Figure 6D). Interestingly, in confocal images of Sf9 cells infected with the PH(111–245)EGFP baculovirus, the EGFP was observed scattered throughout the cell as a soluble protein (Figure 6D). This observation further confirms that the nuclear localization signal of polyhedrin is contained within the first 110 amino acids, as previously suggested , and that the self-aggregating sequence from polyhedra is present within the first 110 amino acids. All these results strongly suggest that only the amino terminus from polyhedrin can be incorporated into polyhedra crystals (when co-expressed with WT polyhedrin).
To further investigate what fragments from the amino terminus of polyhedrin can be incorporated into the polyhedra, we prepared new recombinant baculoviruses containing several fragments from polyhedrin fused to EGFP.
These results suggest the presence of a putative nuclear retention signal shared by fragments 1–58 and 58–110. A previous study identified a nuclear retention signal present within the sequence 1–110 . However, in the aforementioned study they did not test as many polyhedrin fragments as we did in the present study.
To delimit more precisely the minimum amino acid sequence capable of forming canonical polyhedra when co-expressed with WT polyhedrin, we produced several recombinant baculoviruses carrying different fragments from the amino terminus of polyhedrin. We concentrated in the first 58 amino acids, to further delimit the minimal sequence capable of being incorporated into polyhedra.
The fragments PH(1–25)EGFP and PH(25–58)EGFP did not form aggregates, neither they could be incorporated into the polyhedra when co-expressed with WT polyhedrin. Similar results were obtained with the fragment PH(1–34)EGFP, PH(1–48)EGFP, PH(17–58)EGFP or PH(25–49)EGFP. These results indicate that the minimum fragment that can be incorporated into the polyhedra crystal is PH(1–58)EGFP. Similarly, the fragment PH(58–110)EGFP was also incorporated into the polyhedra crystal, but we did not explore other sequences within this later fragment in the present study.
All the results presented in this study show that: a) fragments from the n-terminus of polyhedrin, namely 1–58 and 58–110 can also be incorporated into the canonical polyhedra (when co-expressed with WT polyhedrin), regardless of the cellular localization or the fact that they can self-aggregate (58–110) or not (1–58); b) fragments from the c-terminus of polyhedrin are soluble, found scattered throughout the cell cytosol and nuclei, and are not incorporated into the polyhedra (when co-expressed with WT polyhedrin). In fact, they were completely excluded from the polyhedra crystal, as illustrated in Figure 6D (see also insets D’ and D”).
To obtain quantitative data about the effectiveness of the different fragments to be incorporated into the polyhedra crystal, we conducted flow cytometry studies with polyhedra formed by the combination of the different fragments from polyhedrin fused to EGFP and WT polyhedrin. Polyhedra were purified for these studies prior to conducting the flow cytometry studies (Methods).
Figure 2 shows the results of measuring EGFP fluorescence as reporter of the amount of fragments incorporated into the polyhedra crystals. Figure 2A illustrates typical histograms of single particle fluorescence and Figure 2B summarizes the results (mean ± SD) from at least 3 independent measurements. As illustrated in the figure only fragments PH(1–58)EGFP, PH(58–110)EGFP, PH(1–110)EGFP and the full length PH(1–245)-EGFP were incorporated into polyhedra crystals. Most interestingly, the most fluorescent polyhedra was obtained with the fragment PH(58–110)EGFP, followed by PH(1–110)EGFP and the full length PH(1–245)-EGFP. These results strongly suggest that using the fragment PH(58–110)EGFP results in higher yields of recombinant protein incorporated into the polyhedra crystals.
As we have previously shown, both PH(1–110)EGFP and PH(58–110)EGFP produced electron dense particles identifiable by TEM. Interestingly, both nanoparticles can be isolated from Sf9 cell lysates and retain its structural features. Nanoparticles produced by fragment PH(1–110)EGFP inside Sf9 infected cells are illustrated in Figure 3A. These nanoparticles produced by the self-aggregating PH(1–110)EGFP can be isolated from Sf9 lysates, as illustrated in the electron scanning images from Figure 3B. The nanoparticles have integrated EGFP and the fluorescence is observed by confocal microscopy (Figure 3C). Using an alternative method for nanoparticle size analysis based on Nanoparticle Tracking Analysis (NTA) we identified that both PH(1–110)EGFP and PH(58–110)EGFP produced nanoparticles of about 100 nm in diameter (6D). Notice that the main nanoparticles identified are multiples of 100 nm, which was the most abundant and smallest size identified (6D).
Figure 1A summarizes the fragments that can be incorporated into polyhedra, when co-expressed with WT polyhedrin. Figure 1B illustrates the secondary structure of polyhedrin, obtained from the crystal . As indicated in this figure, polyhedrin is formed of several consecutive β sheets and α helices. The crystallographic study of polyhedrin has identified the first helix (H1) as relevant for the formation of the polyhedrin trimer, which in turn forms the basic cell of the crystal [10,11].
Our studies have identified the fragment PH(58–110)EGFP as containing the sequence/structure essential for the self-aggregation properties of polyhedra (Figure 1C). Using this fragment results in self-aggregated particles scattered in the nuclei and cytosol of infected cells (Figures 1C and 7).
In spite of the fact that both minimal fragments that can be incorporated into polyhedra (PH(1–58)EGFP and PH(58–110)EGFP) when co-expressed with WT polyhedrin have similar structural features: both are formed by two consecutive β sheets followed by a α helix (Figure 1B), only the fragment PH(58–110)EGFP retains the self-aggregation property of polyhedra.
All the results gathered in this study indicate that the sequence/structure required for a fragment to be incorporated into the polyhedra crystal are present in both fragments PH(1–58)EGFP and PH(58–110)EGFP. For this reason the fragment PH(1–110)EGFP is also incorporated into polyhedra when co-expressed with WT polyhedrin. The fragment PH(111–245)EGFP from the C-terminal region of polyhedrin was excluded from the polyhedra crystals (Figure 6D). Noteworthy, not all fragments were incorporated into polyhedra with the same efficiency, the most effective appeared to be PH(58–110)EGFP (Figure 2B). This strongly suggest that the property that facilitates association to WT polyhedrin to form the crystal is in both fragments (PH(1–58)EGFP and PH(58–110)EGFP), but the self-aggregation property is present exclusively in fragment PH(58–110)EGFP. The sequence/structure required for nuclear localization appear to be a combination of fragments PH(1–58)EGFP and PH(58–110)EGFP, since only the fragment PH(1–110)EGFP showed exclusive nuclear localization.
The baculovirus expression system has become a powerful tool for recombinant eukaryotic gene expression . The initial production of recombinant proteins, directed under the strong promoter of polyhedrin, was carried out in cultured cells as soluble proteins. Several modifications have been made over the last ten years to improve the system in order to obtain larger protein yields or adequate the recombinant protein for mammalian usage . Some of these improvements include enhanced trafficking, folding and glycosilation, as well as preventing intracellular degradation. Another recently developed strategy has been the expression of foreign proteins incorporated into the polyhedra crystal . Several proteins have been now incorporated into polyhedra, by fusing them to the full length polyhedrin in three different insect viruses .
Jarvis et al. fused different fragments of AcMNPV polyhedrin to two nonnuclear reporter proteins, β-galactosidase and β-glucorinadase to define its possible nuclear localization signal, and study the distribution and assembly of polyhedra by indirect immunofluorescence and by biochemical fractionation and SDS-PAGE of infected Sf9 . The recombinant proteins produced diffuse or occlusion-like (self-aggregates) proteins, but none of them assembled into the polyhedra.
More recently, Je et al. using GFP fused to AcMNPV polyhedrin, observed the GFP in a soluble form in the nucleus and cytoplasm of infected cells . Only when co-expressed with WT polyhedrin resulted in the formation of polyhedra. Using the elucidated crystal structure of AcMNPV polyhedra we constructed several baculovirus expressing the fluorescent protein EGFP fused to AcMNPV polyhedrin or to its different fragments in order to follow the formation of recombinant polyhedra by confocal microscopy or TEM in Sf9 infected cells.
The N-terminal from 1–110 amino acids of AcMNPV polyhedrin can be divided into two similar fragments (1–57 and 58–110) each of them consisting of two β helices (βA, βA’, and βA”, βB ), followed by a short α helix (α1 and α2) Figure 1B and .
Because the 110 amino acids N-terminal region contributes to the clamping of the 3 molecules of polyhedrin in the trimer (the core of the crystal, ), we considered that as the fragment 1–110 of AcMNPV, these two smaller fragments (1–58 and 58–110) could associate and direct the incorporation of foreign proteins into polyhedra. Analyzing EGFP expression through confocal microscopy and TEM, allowed us to determine the localization and formation of polyhedra in infected Sf9 cells with different polyhedrin fragments.
We observed that the fragments PH(1–58), PH(58–110) and PH(1–110) of AcMNPV can be incorporated into polyhedra, only when co-expressed with the WT polyhedrin. The fact that polyhedra located in the nucleus is observed only when co-expressing WT and recombinant fragments of polyhedrin, suggest that the WT and the different N-terminal fragments identified in this study associate in the cytosol and travel, assembled with WT polyhedrin, to the nucleus of the infected cell. In agreement with this hypothesis, a previous study demonstrated that the nuclear localization of polyhedrin becomes more evident during the phase of occlusion due to the high rate of polyhedrin localization signal and a higher rate of protein biosynthesis .
An important finding in the formation of polyhedra was the ratio of the recombinant fused polyhedrin fragments to the WT polyhedrin. Because the minimal unit of polyhedra crystals is a trimer, the association of the recombinant protein could be carried out in a stoichiometric ratio of 3 WT versus 1 recombinant polyhedrin (Figure 5A), and this parameter could be very important for the formation and quantity of foreign protein that could be incorporated into the crystal.
Noteworthy, not all fragments that incorporate into polyhedra result in equal amounts of fluorescence emitted by the EGFP incorporated into the particles. Most notably the fragment PH(58–110)EGFP produced the brightest particles in our study, as assayed by flow cytometry, followed by fragments PH(1–110)EGFP and PH(1–58)EGFP, in that order. Most surprisingly, the less efficient was the full length polyhedrin (PH(1–245)-EGFP, Figure 2B). Because the brightness of EGFP may reflect also geometric properties of the crystals formed by the different polyhedrin fragments, we conducted western blot analysis of the purified polyhedra formed by the combination of WT polyhedrin and the fragments presented in this study. In general agreement with the flow cytometry results, western blot analysis indicated different ratios of recombinant and WT polyhedrin present in the polyhedra particles (data not shown). These results strongly suggest a different incorporation efficiency for the different fragments studied here, pointing to PH(58–110)EGFP as the most efficient of all. The reasons behind the increased incorporation of PH(58–110)EGFP remains unsolved but are a target of a current study. Most interestingly, this was the minimum fragment that could form self-aggregates, thus a hint about the greater efficiency of incorporation into polyhedra may reside here.
The identification of the amino acids 58–110 as the smallest fragment capable of self-aggregation and efficient incorporation into polyhedra crystals from the N-terminal of AcMNPV is important in the development of new technologies to produce nanoparticles of interest in science and biotechnology, carrying foreign proteins of interest. Most interestingly, the self-aggregates produced by fragments PH(1–110) and PH(58–110) are electron dense particles, as identified in our TEM studies (Figures 3A and 6C), and they could be purified by low speed centrifugation as nanoparticles (Figure 3B-C). These later findings point to the use of fragment PH(58–110) as a powerful tool to produce high yields of recombinant protein that can be easily isolated by slow speed centrifugation from Sf9 cell lysates.
Using a deletion strategy based on the crystallographic structure of AcMNPV polyhedrin protein, we have identified a minimum sequence (PH(58–110)) consisting of two beta domains followed by an alpha helix, which contains a self aggregating domain essential for polyhedra-like particle formation. This deletion strategy allowed also the identification of a nuclear retention signal in polyhedrin, contained within the first 110 amino acids. Even though the fragment PH(1–58) contains also two beta domains followed by an alpha helix, is not sufficient for self-aggregation, since the expression of this fragment results a soluble form contained in the cytosol of infected cells.
These findings open new avenues to explore how polyhedra crystals are formed, and to understand what structural features may be required for in vivo protein crystallization.
This work was supported by grants from Consejo Nacional de Ciencia y Tecnología (CONACyT), Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF) and Dirección General de Asuntos del Personal Académico (DGAPA) to LV. We would like to thank to Dr. Angelica Zepeda for critical reading of our manuscript and suggestions.
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