Development of a Dual-Vector System Utilizing MicroRNA Mimics of the Autographa californica miR-1 for an Inducible Knockdown in Insect Cells.

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years, several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study, an inducible knockdown system was established in insect (Sf9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next, recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting Sf9 cells with these two viruses, the fluorescence intensity of eYFP was suppressed by ~30⁻40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform.

The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation.

Fibroblast growth factor receptor 3 isoforms: Novel therapeutic targets for hepatocellular carcinoma?

UNLABELLED: Fibroblast growth factor receptors (FGFRs) are frequently up-regulated in subsets of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight that FGFR3 splice variants IIIb and IIIc impact considerably on the malignant phenotype of HCC cells. The occurrence of FGFR3 variants was analyzed in human HCC samples. In hepatoma/hepatocarcinoma cell lines, FGFR3 isoforms were overexpressed by lentiviral constructs or down-modulated by small interfering RNA (siRNA; affecting FGFR3-IIIb and -IIIc) or an adenoviral kinase-dead FGFR3-IIIc construct (kdFGFR3). Elevated levels of FGFR3-IIIb and/or -IIIc were found in 53% of HCC cases. FGFR3-IIIb overexpression occurred significantly more often in primary tumors of large (pT2-4) than of small size (pT1). Furthermore, one or both isoforms were enhanced mostly in cases with early tumor infiltration and/or recurrence at the time of surgery or follow-up examinations. In hepatoma/hepatocarcinoma cells, up-regulated FGFR3-IIIb conferred an enhanced capability for proliferation. Both FGFR3-IIIb and FGFR3-IIIc suppressed apoptotic activity, enhanced clonogenic growth, and induced disintegration of the blood/lymph endothelium. The tumorigenicity of cells in severe combined immunodeficiency mice was augmented to a larger degree by variant IIIb than by IIIc. Conversely, siRNA targeting FGFR3 and kdFGFR3 reduced clonogenicity, anchorage-independent growth, and disintegration of the blood/lymph endothelium in vitro. Furthermore, kdFGFR3 strongly attenuated tumor formation in vivo. CONCLUSIONS: Deregulated FGFR3 variants exhibit specific effects in the malignant progression of HCC cells. Accordingly, blockade of FGFR3-mediated signaling may be a promising therapeutic approach to antagonize growth and malignant behavior of HCC cells.

An HER2-Displaying Virus-Like Particle Vaccine Protects from Challenge with Mammary Carcinoma Cells in a Mouse Model.

Human epidermal growth factor receptor-2 (HER2) is upregulated in 20% to 30% of breast cancers and is a marker of a poor outcome. Due to the development of resistance to passive immunotherapy with Trastuzumab, active anti-HER2 vaccination strategies that could potentially trigger durable tumor-specific immune responses have become an attractive research area. Recently, we have shown that budded virus-like particles (VLPs) produced in Sf9 insect cells are an ideal platform for the expression of complex membrane proteins. To assess the efficacy of antigen-displaying VLPs as active cancer vaccines, BALB/c mice were immunized with insect cell glycosylated and mammalian-like glycosylated HER2-displaying VLPs in combination with two different adjuvants and were challenged with HER2-positive tumors. Higher HER2-specific antibody titers and effector functions were induced in mice vaccinated with insect cell glycosylated HER2 VLPs compared to mammalian-like glycosylated counterparts. Moreover, insect cell glycosylated HER2 VLPs elicited a protective effect in mice grafted with HER2-positive mammary carcinoma cells. Interestingly, no protection was observed in mice that were adjuvanted with Poly (I:C). Here, we show that antigen-displaying VLPs produced in Sf9 insect cells were able to induce robust and durable immune responses in vivo and have the potential to be utilized as active cancer vaccines.

Comparative transcriptome analysis of a Trichoplusia ni cell line reveals distinct host responses to intracellular and secreted protein products expressed by recombinant baculoviruses.

The baculovirus insect cell expression system has become a firmly established production platform in biotechnology. Various complex proteins, multi-subunit particles including veterinary and human vaccines are manufactured with this system on a commercial scale. Apart from baculovirus infected Spodoptera frugiperda (Sf9) cells, the Trichoplusia ni (HighFive) cell line is alternatively used as host organism. In this study, we explored the protein production capabilities of Tnms42 insect cells, a new derivative of HighFive, which is free of latent nodavirus infection. As a model system, a cytosolic (mCherry) and a secreted (hemagglutinin) protein were overexpressed in Tnms42 cells. The response of the host cells was followed in a time course experiment over the infection cycle by comparative transcriptome analysis (RNA-seq). As expected, the baculovirus infection per se had a massive impact on the host cell transcriptome, which was observed by the huge total number of differentially expressed transcripts (>14,000). Despite this severe overall cellular reaction, a specific response could be clearly attributed to the overexpression of secreted hemagglutinin, revealing limits in the secretory capacity of the host cell. About 400 significantly regulated transcripts were identified and assigned to biochemical pathways and gene ontology (GO) categories, all related to protein processing, folding and response to unfolded protein. The identification of relevant target genes will serve to design specific virus engineering concepts for improving the yield of proteins that are dependent on the secretory pathway.