Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1.

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Identification of Spodoptera frugiperda importin alphas that facilitate the nuclear import of Autographa californica multiple nucleopolyhedrovirus DNA polymerase.

Proteins containing nuclear localization signals (NLSs) are actively transported into the nucleus via the classic importin-α/ß-mediated pathway, and NLSs are recognized by members of the importin-α family. Most studies of insect importin-αs have focused on Drosophila to date, little is known about the importin-α proteins in Lepidoptera insects. In this study, we identified four putative importin-α homologues, Spodoptera frugiperda importin-α1 (SfIMA1), SfIMA2, SfIMA4 and SfIMA7, from Sf9 cells. Immunofluorescence analysis showed that SfIMA2, SfIMA4 and SfIMA7 localized to the nucleus, while SfIMA1 distributed in cytoplasm. Additionally, SfIMA4 and SfIMA7 were also detected in the nuclear membrane of Sf9 cells. SfIMA1, SfIMA4 and SfIMA7, but not SfIMA2, were found to associate with the C terminus of AcMNPV DNA polymerase (DNApol) that harbours a typical monopartite NLS and a classic bipartite NLS. Further analysis of protein-protein interactions revealed that SfIMA1 specifically recognizes the bipartite NLS, while SfIMA4 and SfIMA7 bind to both monopartite and bipartite NLSs. Together, our results suggested that SfIMA1, SfIMA4 and SfIMA7 play important roles in the nuclear import of AcMNPV DNApol C terminus in Sf9 cells.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving ∆11 Desaturation and an Elusive ∆7 Desaturase.

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

High-volume shake flask cultures as an alternative to cellbag technology for recombinant protein production in the baculovirus expression vector system (BEVS).

Recombinant protein production in the baculovirus expression vector system (BEVS) has emerged as a system of choice for the production of recombinant human proteins for R&D purposes. Scale-up protein production in insect cells past the one or two liter volume generally utilizes disposable cellbag bioreactors that provide a means to scale to the 5-25L range in a single vessel. However, cellbags can be expensive and their use requires capital investment in dedicated rocker platforms and their associated air pumps and exhaust heaters. Additional equipment, such as tube welders and liquid pumps are often also deployed for the sterile transfer of media outside of a biosafety cabinet. Herein it is reported that Sf9, Sf21 and High Five insect cells demonstrate normal growth characteristics when cultured at the 2.5 L level in 3 L Erlenmeyer flasks, or at the 4.5 L level in 5 L Erlenmeyer flasks in standard laboratory shakers. In addition, a direct comparison of the expression levels of four separate proteins at the 4.5 L scale in 5 L flasks versus those at the 5 L scale in 10 L cellbags demonstrates that protein production is equal to, or slightly better, in the flasks versus the cellbags. The adoption of high-volume shake flasks for routine recombinant protein production in insect cells has a number of advantages over disposable bioreactors in terms of ease of use, and equipment and disposables costs.

Purification of metal-dependent lysine deacetylases with consistently high activity.

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

Secretory expression and scale-up production of recombinant human thyroid peroxidase via baculovirus/insect cell system in a wave-type bioreactor.

The human thyroid peroxidase (hTPO) is an essential enzyme for thyroid hormone biosynthesis and is expressed in thyroid cells. It is an autoantigen against which antibodies are found in the sera of patients with a number of autoimmune thyroid disorders. Overexpression of hTPO has been achieved using the baculovirus expression vector system (BEVS). However, it is produced largely in an aggregated form in the cell lysate fraction, which increases the complexity of protein extraction. In this study, to achieve improved secretory expression of hTPO via BEVS, a truncated recombinant hTPO protein (hTPOt) was engineered by replacing its original signal peptide (SP) in the N-terminal with five heterologous SPs. Our data showed that the SP from the peptidyl-glycine alpha-amidating monooxygenase (PAM), referred to as SPPAM, significantly promoted the secretion of SPPAM-fused hTPOt (p-hTPOt) in High Five cells. Subsequently, we established an optimized scale-up production procedure for p-hTPOt in a 5-L wave-type bioreactor. The secretory p-hTPOt was purified by immobilized metal-chelating affinity chromatography and ion-exchange chromatography, achieving a protein purity of >95%. Finally, the purified p-hTPOt showed high sensitivity and specificity in reactions with positive or negative human serum samples via the double-antigen sandwich method, suggesting potential applications in hTPO-based research and product development.

Transcriptome analysis of Spodoptera frugiperda 9 (Sf9) cells infected with baculovirus, AcMNPV or AcMNPV-BmK IT.

OBJECTIVES: To analyze the transcriptome of Spodoptera frugiperda 9 (Sf9) cells infected with AcMNPV or AcMNPV-BmK IT. RESULTS: A comprehensive transcriptome profile for Sf9 cells infected with AcMNPV or AcMNPV-BmK IT is shown. 43127572, 46529744 and 47235310 RNA-Seq profiles permitted the quantification of expression levels for control (C), AcMNPV-BmK IT treatment (ABT) and AcMNPV treatment (AT) groups. There were 997 up-regulated or down-regulated candidate genes with significant different expression level in these treatment groups. CONCLUSION: These results provide a broad spectrum of candidate genes that are critically involved in the molecular regulation mechanism of Sf9 cells infected with AcMNPV-BmK IT.

Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity.

Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a racemate of R- and S-isomers, and CYP2A6 plays an important role in the enantioselective metabolism of FT in human liver microsomes (R-FT >> S-FT). This study examined the enantioselective metabolism of FT by microsomes prepared from Sf9 cells expressing wild-type CYP2A6 and its variants (CYP2A6*7, *8, *10, and *11) that are highly prevalent in the Asian population. We also investigated the metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in these variants. Enzyme kinetic analyses showed that CYP2A6.7 (I471T) and CYP2A6.10 (I471T and R485L) had markedly lower Vmax values for both enantiomers than wild-type enzyme (CYP2A6.1) and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both enantiomers than CYP2A6.1. The ratios of Vmax and Km values for R-FT to corresponding values for S-FT (R/S ratio) were similar among enzymes, indicating little difference in enantioselectivity among the wild-type and variant enzymes. Similarly, both CYP2A6.7 and CYP2A6.10 had markedly lower Vmax values for coumarin 7-hydroxylase and nicotine C-oxidase activities than CYP2A6.1 and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both activities than CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6 variants generally resulted in lower affinity for substrates, while Vmax values were selectively reduced in CYP2A6.7 and CYP2A6.10. Consistent R/S ratios among CYP2A6.1 and variant enzymes indicated that the amino acid substitutions had little effect on enantioselectivity in the metabolism of FT.

Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems.

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: ß-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.

Component co-expression and purification of recombinant human pyruvate dehydrogenase complex from baculovirus infected SF9 cells.

The mammalian pyruvate dehydrogenase complex (PDC) is a multi-component mitochondrial enzyme that plays a key role in the conversion of pyruvate to acetyl-CoA connecting glycolysis to the citric acid cycle. Recent studies indicate that targeting the regulation of PDC enzymatic activity might offer therapeutic opportunities by inhibiting cancer cell metabolism. To facilitate drug discovery in this area, a well defined PDC sample is needed. Here, we report a new method of producing functional, recombinant, high quality human PDC complex. All five components were co-expressed in the cytoplasm of baculovirus-infected SF9 cells by deletion of the mitochondrial localization signal sequences of all the components and E1a was FLAG-tagged to facilitate purification. The protein FLAG tagged E1a complex was purified using FLAG-M2 affinity resin, followed by Superdex 200 sizing chromatography. The E2 and E3BP components were then Lipoylated using an enzyme based in vitro process. The resulting PDC is over 90% pure and homogenous. This non-phosphorylated, lipoylated human PDC was demonstrated to produce a robust detection window when used to develop an enzyme coupled assay of PDHK.

The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1.

Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.

Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of ß-catenin.

ß-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although ß-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of ß-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular ß-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of ß-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of ß-catenin within this complex. Each component of ß-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential ß-catenin-interacting proteins, and define when and where a specific complex forms.

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells.

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2'-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Process intensification for the continuous production of an antimicrobial peptide in stably-transformed Sf-9 insect cells.

The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical technologies such as turbidimetry and dielectric spectroscopy. We developed batch and perfusion processes at the 2-L scale for the production of BR033, a cecropin-like AMP from Lucilia sericata, in stably-transformed polyclonal Sf-9 cells. This is the first time that BR033 has been expressed as a recombinant peptide. Process analytical technology facilitated the online monitoring and control of cell growth, viability and concentration. The perfusion process increased productivity by ~ 180% compared to the batch process and achieved a viable cell concentration of 1.1 × 107 cells/mL. Acoustic separation enabled the consistent retention of 98.5-100% of the cells, viability was > 90.5%. The recombinant AMP was recovered from the culture broth by immobilized metal affinity chromatography and gel filtration and was able to inhibit the growth of Escherichia coli K12. These results demonstrate a successful, integrated approach for the development and intensification of a process from cloning to activity testing for the production of new biopharmaceutical candidates.

Development of a non-viral platform for rapid virus-like particle production in Sf9 cells.

Insect cells have shown a high versatility to produce multiple recombinant products. The ease of culture, low contamination risk with human pathogens and high expression capacity makes an attractive platform to generate virus-like particles (VLPs). The baculovirus expression vector system (BEVS) has been frequently used to produce these complex nanoparticles. However, the BEVS entails several difficulties in the downstream phase as well as undesirable side-effects due to the expression of baculovirus-derived proteins. In this work, we developed a baculovirus-free system based on polyethylenimine (PEI)-mediated transient gene expression (TGE) of Sf9 cells. An exhaustive study of DNA:PEI polyplex formation was performed and the optimal TGE conditions were determined by the combination of Design of Experiments (DoE) and desirability functions. The TGE approach was successfully applied to produce three model recombinant products with different structural complexities, including eGFP, hSEAP and HIV-1 Gag VLPs. Cell membrane co-localization with the Gag polyprotein was detected by fluorescence microscopy, whereas nanoparticle tracking analysis and flow virometry were applied as high-throughput techniques to monitor the VLP production process. Analysis of VLP production revealed that 48 h after transfection were optimal for VLP harvesting since the ratio of VLPs to extracellular vesicles was the highest. In these conditions, a maximum of 1.9 ±â€¯0.8·109 VLP/mL was achieved, representing a 2.8-fold increase compared to the initial transfection condition. In conclusion, the TGE approach proposed in this study provides a baculovirus-free platform to rapidly produce VLPs and potentially other recombinant products in insect cells.

Dioleoylphosphatidylglycerol Accelerates Corneal Epithelial Wound Healing.

Purpose: In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. Methods: We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. Results: AQP3 was present in human corneas in situ, and AQP3 and PLD2 were co-immunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3- and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). Conclusions: These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1).

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

Spinosad induces programmed cell death involves mitochondrial dysfunction and cytochrome C release in Spodoptera frugiperda Sf9 cells.

Spinosad, a reduced-risk insecticide, acts on the nicotinic acetylcholine receptors and the gamma-aminobutyric acid receptor in the nervous system of target insects. However, its mechanism of action in non-neural insect cells is unclear. This study aimed to evaluate mitochondrial functional changes associated with spinosad in Spodoptera frugiperda (Sf9) insect cells. Our results indicate that in Sf9 cells, spinosad induces programmed cell death and mitochondrial dysfunction through enhanced reactive oxygen species production, mitochondrial permeability transition pore (mPTP) opening, and mitochondrial membrane potential collapse, eventually leading to cytochrome C release and apoptosis. The cytochrome C release induced by spinosad treatment was partly inhibited by the mPTP inhibitors cyclosporin A and bongkrekic acid. Subsequently, we found that spinosad downregulated Bcl-2 expression and upregulated p53 and Bax expressions, activated caspase-9 and caspase-3, and triggered PARP cleavage in Sf9 cells. These findings suggested that spinosad-induced programmed cell death was modulated by mitochondrial dysfunction and cytochrome C release.

Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli.

Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017.