Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.

Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system.

The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or ß-synuclein (ß-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, ß-syn + CALR, or ß-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three "helper" genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three "helper" genes, up to 10 dpi was observed. Utilization of this "triple-supporters" containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products.

Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2µg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2µg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC.

Α-synuclein and ß-synuclein enhance secretion protein production in baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of α-synuclein (α-syn) and ß-synuclein (ß-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that α- and ß-syn can enhance secretion proteins production in BEVS.

Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E.

Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase-EGFP fusion protein (SEFP) can act as a traceable and sensitive secretory reporter protein in BEVS. In this study, a chaperone, calreticulin (CALR), and the translation initiation factor eIF4E were co-expressed with SEFP using a bicistronic baculovirus expression vector. We observed that the intracellular distribution of SEFP in cells co-expressing CALR was different from co-expressing eIF4E. The increased green fluorescence emitted by cells co-expressing CALR had a good correlation with the abundance of intracellular SEFP protein and an unconventional ER expansion. Cells co-expressing eIF4E, on the other hand, showed an increase in extracellular SEAP activity compared to the control. Utilization of these baculovirus expression constructs containing either eIF4E or CALR offers a significant advantage for producing secreted proteins for various biotechnological and therapeutic applications.

A bi-cistronic baculovirus expression vector for improved recombinant protein production.

Baculoviruses are one of the most studied insect viruses both in basic virology research and in biotechnology applications. Incorporating an internal ribosome entry site (IRES) into the baculovirus genome generates bi-cistronic baculoviruses expression vectors that produce two genes of interest. The bi-cistronic baculoviruses also facilitate recombinant virus isolation and titer determination when the green fluorescent protein was co-expressed. Furthermore, when the secretion proteins were co-expressed with the cytosolic green fluorescent protein, the cell lysis and cytosolic protein released into the culture medium could be monitored by the green fluorescence, thus facilitating purification of the secreted proteins.

Functional expression of rat neuroligin-1 extracellular fragment by a bi-cistronic baculovirus expression vector.

The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. Here, we describe the simultaneous expression of the extracellular domain of rat neuroligin-1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant rat NL1 protein, fused with signal sequence derived from human Azurocidin gene (AzSP), was secreted into the culture medium and the optimum harvest time for NL1 protein before the lysis of infected cells was determined through the release of cytosolic EGFP. The NL1 protein (0.129±0.013 mg/8×10(7) High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified rat NL1 promoted and enhanced the growth rate (137.07±9.74 µm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53±4.53 µm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies.

Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.

Recombinant baculoviruses are suitable for the high-level production of large multi-protein complexes. A tri-cistronic expression vector was constructed by the inclusion of two internal ribosome entry sites (IRESs). In this novel polycistronic vector, one single polyhedrin promoter controlled the transcription of a tri-cistronic transcript. Also, the first cistron was translated through a cap-dependent mechanism, while the second and third cistrons were translated by the IRESs derived from the 5' UTR of Rhopalosiphum padi virus (RhPV) and Perina nuda virus (PnV), respectively. The ratio of tri-cistronic gene expression levels produced by the three translational initiation modules is about 2:1:1 (cap:PnV IRES:RhPV IRES). This study indicates that polycistronic genes can be co-expressed at the translational level as in prokaryotic expression system by baculovirus biotechnology.

Internal ribosome entry site of Rhopalosiphum padi virus is functional in mammalian cells and has cryptic promoter activity in baculovirus-infected Sf21 cells.

AIM: To substantiate the in vitro translational studies of a cross-kingdom, internal ribosome entry site (IRES), the 5 untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells. METHODS: Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to test whether the RhPV IRES can mediate translation activity in versatile mammalian cell lines. RESULTS: Both plasmids and recombinant baculoviruses containing the CMV promoter and the RhPV IRES can mediate bicistronic gene expression in mammalian cells. However, in the CMV promoter containing recombinant baculovirus-infected insect Sf21 cells, only the second cistron gene expression was observed. Northern blot analysis and a promoterless assay demonstrated that the RhPV IRES exhibited cryptic promoter activity in baculovirus-infected insect cells. CONCLUSION: RhPV IRES can mediate gene expression in both insect cells and mammalian cells, and this characteristic of the RhPV IRES will facilitate the development of a bicistronic baculovirus gene therapy vectors.

Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.