[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Protein Expression and Purification 09/2011; 81(1):18-24. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral 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, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion.
Journal of virological methods 08/2011; 175(2):206-15. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 µg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.
Bioscience Biotechnology and Biochemistry 07/2011; 75(7):1342-8. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of virological methods 09/2009; 159(2):152-9. · 2.13 Impact Factor