Comparison between different conditions for the incorporation of foreign proteins into Autographa californica multiple polyhedrovirus polyhedra for biotechnological purposes.

The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.

Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC).

Hyperexpression of polh and p10, two very late genes, is one of the remarkable characteristics in the baculovirus life cycle. However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC-MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).

Molecular mechanism responsible for the hyperexpression of baculovirus polyhedrin.

One of the amazing phenomena in the baculovirus life cycle is the hyperexpression of the very late gene, polyhedrin (polh), causing the production of the occlusion bodies where progeny virions are embedded. However, to date, the molecular mechanism underlying its hyperexpression is not completely elucidated. Considering that, in this review, the mechanism responsible for its hyperexpression from the previous studies up to now was comprehensively summarized from three aspects, namely, the structure characteristics of the polh promoter and transcription regulation, the structure and translation regulation of the polh mRNA, and especially the regulators that influence the expression of polh gene. Moreover, this review will help us obtain a better understanding about the hyperexpression of polh, and also provide guidance for improving the expression efficiency of the foreign proteins by adopting the baculovirus expression vector system.

Serial protein crystallography in an electron microscope.

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

A self-aggregating peptide: implications for the development of thermostable vaccine candidates.

BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.

New method for immobilising diverse proteins onto cubic micro-protein polyhedrin crystals.

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

A simplified strategy to package foreign proteins into baculovirus occlusion bodies without engineering the viral genome.

Polyhedron envelope protein (PEP) is the major component of the calyx that surrounds the baculovirus occlusion body (OB). PEP has been associated with the stabilization and resistance of polyhedra in the environment. Due to the abundant levels of PEP in OBs, we decided to use this protein as a fusion partner to redirect foreign proteins to baculovirus polyhedra. In this study we developed a strategy that involves the generation of a monoclonal transformed insect cell line expressing a protein of interest fused to the the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) N-terminus of PEP that enables the packaging of foreign proteins into the OBs without generating a recombinant baculovirus. This proved to be an efficient platform that could be exploited to improve wild type baculovirus for their use as bioinsecticides without facing the concerns of releasing genetically modified DNA to the environment and bypassing the associated regulatory issues. We demonstrated, using immunological, proteomic and microscopy techniques, that the envelope of AgMNPV OBs can effectively trap chimeric proteins in an infected insect cell line expressing AgMNPV PEP fused to the enhanced green fluorescent protein (eGFP). Furthermore, packaging of chimeric PEP also took place with heterologous OBs such as those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), another group I alphabaculovirus.

Major capsid protein of Autographa californica multiple nucleopolyhedrovirus contributes to the promoter activity of the very late viral genes.

The baculovirus expression vector system (BEVS) is one of the most powerful eukaryotic expression systems. Recombinant protein expression is usually controlled by promoters of the baculovirus very late genes (i.e., polyhedrin and p10); therefore, identifying novel regulatory factors for these promoters is key to increasing BEVS productivity. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the viral vector most frequently used in BEVS. VP39 is the major nucleocapsid protein of AcMNPV and plays a pivotal role in nucleocapsid assembly in the nucleus. In this study, we found that knocking out vp39 from the AcMNPV genome resulted in decreased protein abundance of polyhedrin and P10. Further assays revealed that the mRNA transcripts and the promoter activities of polyhedrin and p10 were decreased in the absence of vp39, suggesting that VP39 contributes to the activity of the very late viral gene promoters and may represent a means of optimizing the current BEVS.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

A Novel Double Core-Shell Bionanoparticle Formed by Ferritin and Baculoviral Polyhedrin.

In this study, we got a step forward of the baculoviral polyhedrin (Polh) exploitation as a new nanobiomaterial. We recruited human H-chain ferritin (FTH) as scaffold and Gaussia luciferase (hGluc) as luminescent signal molecule, preparation, characterization and luminescent analysis of the bionanoparticles formed by the fusion proteins of them mentioned above inspire us to present a view that baculoviral polyhedrin is a nanobrick to be a new nanobiomaterial. The bionanoparticle that formed by FTH and Polh fusion protein is a novel double core-shell structure. The properties of baculoviral polyhedrin make itself a promising new nanobiomaterial for application in bionanotechnology.

Cell-line-dependent crystal morphology and sublocalization of the Thyrinteina arnobia cypovirus polyhedrin expressed from a recombinant baculovirus.

We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 µm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 µm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location.

Biotechnological applications of occlusion bodies of Baculoviruses.

The ability of Baculoviruses to hyper-express very late genes as polyhedrin, the major component of occlusion bodies (OBs) or polyhedra, has allowed the evolution of a system of great utility for biotechnology. The main function of polyhedra in nature is to protect Baculovirus in the environment. The possibility of incorporating foreign proteins into the crystal by fusing them to polyhedrin (POLH) opened novel potential biotechnological uses. In this review, we summarize different applications of Baculovirus chimeric OBs. Basically, the improvement of protein expression and purification with POLH as a fusion partner; the use of recombinant polyhedra as immunogens and antigens, and the incorporation of proteins into polyhedra to improve Baculoviruses as bioinsecticides. The results obtained in each area and the future trends in these topics are also discussed.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations.

Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins.

Polyhedra can encapsulate other proteins and have potential applications as protein stabilizers. The extremely stable polyhedra matrix may provide a platform for future engineered micro-crystal devices. However, the protein composition of the polyhedra matrix remains largely unknown. In this study, the occlusion-derived virus (ODV)-removed BmNPV polyhedra matrix fraction was subjected to SDS-PAGE and then an LC-ESI-MS/MS analysis using a Thermo Scientific Q Exactive mass spectrometer. In total, 28 host and 91 viral proteins were identified. The host components were grouped into one of six categories, i.e., chaperones, ubiquitin and related proteins, host helicases, cytoskeleton-related proteins, RNA-binding proteins and others, according to their predicted Pfam domain(s). Most viral proteins may not be essential for polyhedra assembly, as evidenced by studies in the literature showing that polyhedra formation occurs in the nucleus upon the disruption of individual genes. The structural role of these proteins in baculovirus replication will be of significant interest in future studies. The immobilization of enhanced green fluorescent protein (eGFP) into the polyhedra by fusing with the C-terminus of BM134 that is encoded by open reading frame (ORF) 134 suggested that the polyhedra had a powerful capacity to trap foreign proteins, and BM134 was a potential carrier for incorporating proteins of interest into the polyhedra.

Improved pFastBac™ donor plasmid vectors for higher protein production using the Bac-to-Bac® baculovirus expression vector system.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based Bac-to-Bac® expression system consists of a bacmid and five pFastBac™ donor transfer vectors. It has been widely used for eukaryotic gene expression in insect cells to elucidate gene function in biotechnology laboratories. The pFastBac™ vectors contain a 50bp AcMNPV polyhedrin (polh) promoter and a 127bp SV40 polyadenylation (pA) signal for cloning a gene of interest into the bacmid, resulting in unsolved lower gene expression levels than the wild type (wt) AcMNPV in insect cells. Therefore, the purpose of this research is to understand why the Bac-to-Bac system produces lower gene expression levels. Here, we determined that bacmids transposed with pFastBac™ vectors produced 3-4 fold lower levels of certain proteins than the wt AcMNPV. We found that an 80bp cis element 147bp upstream of the 50bp polh promoter and a 134bp polh pA signal are required in pFastBac™ to achieve bacmid protein expression levels equivalent to wt AcMNPV in High Five insect cells. Therefore, researchers currently using pFastBac™ vectors for protein expression can transfer their genes of interest into the improved vectors in this report to elevate protein expression yields in insect cells to reduce protein production costs.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

Characterization of aggregate/aggresome structures formed by polyhedrin of Bombyx mori nucleopolyhedrovirus.

Virus infections often lead to formation of aggregates and aggresomes in host cells. In this study, production of aggregates and aggresomes by the highly expressed protein polyhedrin of Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h postinfection (p.i.) was detected with a fluorescent molecular dye, and verified by colocalization of polyhedrin with aggresomal markers, GFP-250 and γ-tubulin. Polyhedrin aggregates showed hallmark characteristics of aggresomes: formation was microtubule-dependent; they colocalized with heat shock cognates/proteins of the 70-kDa family (HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria. Aggregated polyhedrin protein gradually gained its active conformation accompanying progress of BmNPV infection. At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production. These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

BACKGROUND: 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. RESULTS: 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 ß 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. CONCLUSIONS: 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.