Baculovirus-based Expression System Research Articles

Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex

Ozz, a member of the SOCS-box family of proteins, is the substrate-binding component of CRL5Ozz, a muscle-specific Cullin-RING ubiquitin ligase complex composed of Elongin B/C, Cullin 5 and Rbx1. CRL5Ozz targets for proteasomal degradation selected pools of substrates, including sarcolemma-associated β-catenin, sarcomeric MyHCemb and Alix/PDCD6IP, which all interact with the actin cytoskeleton. Ubiquitination and degradation of these substrates are required for the remodeling of the contractile sarcomeric apparatus. However, how CRL5Ozz assembles into an active E3 complex and interacts with its substrates remain unexplored. Here, we applied a baculovirus-based expression system to produce large quantities of two subcomplexes, Ozz–EloBC and Cul5–Rbx1. We show that these subcomplexes mixed in a 1:1 ratio reconstitutes a five-components CRL5Ozz monomer and dimer, but that the reconstituted complex interacts with its substrates only as monomer. The in vitro assembled CRL5Ozz complex maintains the capacity to polyubiquitinate each of its substrates, indicating that the protein production method used in these studies is well-suited to generate large amounts of a functional CRL5Ozz. Our findings highlight a mode of assembly of the CRL5Ozz that differs in presence or absence of its cognate substrates and grant further structural studies.

Non-Assembled ORF2 Capsid Protein of Porcine Circovirus 2b Does Not Confer Protective Immunity.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies

BackgroundClostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1–250), and the C-terminal region (aa residues 251–370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms.ResultsIn the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250–363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250–363H6 neutralized the phospholipase C activity of full-length PLC.ConclusionsThe L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Evaluation of theanti-apoptotic activity of bovine alphaherpesvirus type 5 US3 protein kinase in insect cells using a recombinant baculovirus.

Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPV anti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

Potential of Silkworm in Biotechnology

Silkworms have been in use for human needs for centuries. The silk derived from the silkworm is of great economical value. Lately insects and in particular silkworms have attracted the attention for expressing proteins of high value, which need post-translational modifications for human applications or research. At 1985 Dr. Maeda demonstrated that silkworm larvae could produce functional interferon [1]. Since then, to express desired protein of interest in silkworms various techniques have been developed, and among them Baculovirus based expression system (BES) comes foremost. Baculovirus being the ideal vector carrying desired cDNA of interest does not infect nor cause any known disease to humans or other livestock other than its host, thus dismaying any biohazard concerns. Thus it suits the purpose and with the advent of bacmid technology, the ability to produce recombinant baculovirus has grown many folds; both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). BmNPV based expression system for expressing proteins was improvised using BmNPV bacmid [2]. BmNPV bacmid is capable of replicating in both Escherichia coli and expressing protein of interest in Bombyx mori derived cell lines or silkworms. This method utilizes the advantage of a bacmid, it can be easily prepared and screened in E. coli to produce sufficient DNA for subsequent expression in silkworms. Today’s biotechnology based industry faces a bottleneck towards further advancement owing to the lack of proper gene expression systems. In spite of rapid growth of bioscience, there are still many proteins of which structures are unidentified. Scientists concerned in the field of life science desired technique, which can express protein in large quantities, and purify protein in form having a biological function quickly. Thus said, with the completion of Human Genome Project, we have come across a herculean task of trying to understand the function of the proteins being coded by the genes. Many researchers proved silkworm biotechnology is one of solutions. Up to now, α-interferon, HBeAg, sugar-modified eukaryotic proteins, such as carbohydrate transferases and cell surface receptors, have been produced using B. mori silkworm larvae. This work demonstrates great potential advantages of the BmNPV bacmid system for the large-scale expression of foreign genes in the silkworm. Furthermore, recent reports showed that the recombinant baculovirus containing mammalian cell-active promoter can exhibit efficient gene transfer of potential vaccines into mammalian cells. This would be a great breakthrough in production of recombinant eukaryotic proteins and viruses, which will be a powerful tool in a new proteome era. This special issue aims at contributing in the field of life science, for example gene expression system, silkworm research directionality, and commercialized proteins from silkworm expression system, and focusing on genetics of the silkworm, developing new specialized silkworm for protein expression, application of proteins to medicine, transgenic silkworm, and the commercialized proteins in Japan.

Optimization of chimeric HIV‐1 virus‐like particle production in a baculovirus‐insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 x 10(6) cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins.

<I>Glossina</I> Proteolytic Lectin Does Not Require a Carbohydrate Moiety for Enzymatic or Trypanosome-Transforming Activities

The developmental cycle of the cyclically transmitted African trypanosome involves an obligatory passage through the tsetse fly, Glossina spp. This intricate relationship requires the presence of molecules within the insect vector, including a midgut lectin, that interact with the trypanosome. Recently, a gene encoding for a proteolytic lectin, with trypanosome-transforming activity, was isolated from a midgut cDNA library of Glossina fuscipes fuscipes Austen in our laboratory. Using the same approach, we have identified a similar gene from a midgut cDNA library of Glossina austeni (Newstead). The protein encoded by this gene was expressed in bacteria and a baculovirus-based expression system. The baculovirus-expressed lectin was found in the medium of baculovirus-infected Sf-21 cell cultures, indicating that the tsetse fly-derived signal peptide was recognized and cleaved by the Sf-21 cells. The baculovirus-expressed protein also was glycosylated despite the absence of classical O-linked and N-linked sugar attachment motifs. Both the baculovirus- and bacterium-expressed lectin proteins were shown to agglutinate trypanosomes and rabbit red blood cells in vitro. This agglutination was strongly inhibited by D-glucosamine. D-Glucosamine also inhibited the action of the authentic and recombinant lectins upon the chromogenic substrate Chromozym TRY. Interestingly, both baculovirus- and bacterium-expressed lectins showed no significant differences in terms of these activities, indicating that a sugar moiety is not essential for biological activity. Our results provide an important molecular tool for further characterization of Glossina proteolytic lectin.

Construction of hybrib Autographa californica nuclear polyhedrosis bacmid by modification of p143 helicase

We developed a new hybrid nuclear polyhedrosis virus (NPV) bacmid capable of infecting Spodoptera frugiperda, Tricoplusia ni, and Bombyx mori, and B. mori cell lines for producing hybrid recombinant baculovirus that can carry a gene of interest and express it in a broad range of hosts. A GFP uv-β1,3- N-acetylglucosaminyltransferase 2 fusion gene was expressed successfully in silkworm larvae using this hybrid bacmid. The hybrid NPV bacmid provides an altogether simple and realistically feasible method for large-scale applications using silkworm larvae. It can be easily managed in E. coli, which has no biohazard safety concerns, in addition to the baculovirus-based expression system.

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.

Heterologous expression of maize ( Zea mays L.) Mir1 cysteine proteinase in eukaryotic and prokaryotic expression systems

Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

Baculovirus-insect cell expression, purification, and immunological studies of the full-length Japanese encephalitis virus envelope protein

The E protein of Japanese encephalitis virus (JEV) is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. In this study, we developed a baculovirus-based expression system of the Japanese encephalitis virus full-length E protein for production of recombinant vaccine. The expression of the full-length JEV E protein was not secreted but produced in the cytoplasm in spite of the use of prM-derived signal peptide. The full-length JEV E protein expressed in insect cells was extensively purified using immobilized metal affinity chromatography and emulsified with Freund’s adjuvant for immunological studies. Our studies demonstrated the mice immunized with the full-length JEV E protein successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. This finding provides valuable information for developing subunit vaccines for JEV antigenic complex viruses.

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus-infected insect cells. This 21 bp sequence element is derived from a 5′ untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20- and seven-fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A-rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus-based expression system.

Expression of human collagenase I (MMP-1) and TIMP-1 in a baculovirus-based expression system.

Expression of human collagenase I (MMP-1) and TIMP-1 in a baculovirus-based expression system.

Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4 3(1)2 12 with cell constants of a = b = 105.6 A ̊ , c = 506.9 A ̊ . The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of α-helical coiled-coil. Thus in the region of 10-5.5 Å resolution along the c ∗- axis , the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 Å along the c ∗- axis , whereas along the a ∗–b ∗- plane reflections were limited to 6.6 Å. Data analysis is under way on a data set from a PtCl 4 derivative.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-CIS retinol dehydrogenase.

Identification of a 32-kd protein in the bovine retinal pigment epithelium.A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated.The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity.Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture

The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvoifia serpentina and the berberine bridge enzyme [( S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.

A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding.

The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.

Baculovirus expression of antibodies: A method for the expression of complete immunoglobulins in a eukaryotic host

To express complete immunoglobulin heterodimers in vitro, we have taken advantage of the baculovirus expression system. Several approaches to the simultaneous expression of recombinant heavy and light chains were examined, including coinfection with separate γ and κ expressing viruses, as well as infection with a single double recombinant virus that expresses both polypeptides. In both cases, the antibodies produced were correctly processed, glycosylated, and assembled into normal immunoglobulin heterodimers. Furthermore, the antibodies recovered from the supernatants of infected cultures behaved indistinguishably from B-cell-derived immunoglobulins with similar specificities. An additional benefit of a baculovirus-based expression system is the high yield of recombinant material produced in insect cells. Thus we describe here a general method for the high-level expression of authentic antibodies in a eukaryotic host.