A new technique for producing recombinant baculovirus directly in silkworm larvae

A new technique for the direct production of recombinant baculovirus in the silkworm larvae is described. To assess the utility of this method, a combination of Bombyx mori nucleopolyhedroviral genome, transfer vector and Lipofectin was co-injected directly into newly ecdysed fifth instar, silkworm larvae. The recombinant virus was obtained from the hemolymph of injected larvae and the hemolymph then re-injected into the larvae as an inoculum. This resulted in a high-level production of foreign protein in the silkworm larvae. This technique produces easy and rapid recombinant protein production in silkworms.     

High level expression of a frog α-amidating enzyme,AE-II,in cultured cells and silkworm larvae using aBombyx mori nuclear polyhedrosis virus expression vector

AXenopus laevis peptidyl C-terminal α-amidating enzyme (AE-II) gene, modified by deletion of a region encoding the putative membrane-spanning domain and the putative C-terminal cytosolic tail, was expressed in BoMo-15 AIIc insect cells and silkworm larvae using aBombyx mori baculovirus expression vector system. The expressed enzyme was identified predominantly in the culture medium and the hemolymph of silkworm larvae, indicating successful secretion of the expressed AE-II. The level of recombinant enzyme in the larval hemolymph at 4 days post-infection (40 μg/ml) was more than 100-fold the peak levels found in the culture medium (250 ng/ml). The enzyme activity in the larval hemolymph at 4 days post-infection was 3700 units/ml.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Molecular Chaperone-Assisted Production of Human α-1,4-<Emphasis Type="Italic">N</Emphasis>-Acetylglucosaminyltransferase in Silkworm Larvae Using Recombinant BmNPV Bacmids

In this study, human α-1,4-N-acetylglucosaminyltransferase (α4GnT) fused with GFP_uv (GFP_uv-α4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFP_uv-α4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of α4GnT activity in suspension culture. When Bombyx mori nucleopolyhedrovirus containing a GFP_uv-α4GnT fusion gene (BmNPV-CP ⁻/GFP_uv-α4GnT) bacmid was injected into silkworm larvae, α4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher than that of the Tn-pXgp-GFP_uv-α4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under the control of the ie-2 promoter, α4GnT activity in larval hemolymph increased by 1.4–2.0-fold. Moreover, when BmNPV-CP ⁻/GFP_uv-α4GnT bacmid injection was delayed for 3 h after BmNPV-CP ⁻/CNX injection, the α4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFP_uv-α4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV bacmid is a promising tool for recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins.     

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.     

Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.     

Comparison of the efficiencies of different affinity tags in the purification of a recombinant secretory protein expressed in silkworm larval hemolymph

Silkworms are useful bioreactors for heterologous protein expression when used in conjunction with the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. However, purification from silkworm hemolymph is difficult since it contains various kinds of proteins. In this study, we investigated an effective single-step method for the purification of affinity-tagged single-chain antibody variable region fragment (scFv) from silkworm larval hemolymph. A 5-fold higher expression level was obtained when scFv was fused with the His tag than when it was fused with the Strep II or GST tags. However, the His tag was inadequate for single-step purification since it led to the nonspecific binding of contaminants. The purification recoveries of GST-, Strep II-, and His-tagged scFvs were 91.8%, 43.7%, and 27.2%, respectively. The specific amount of single-step purified GST-tagged scFv was 2.2∼2.7 fold higher than the amounts of the His- and Strep II-tagged constructs. The purities of Strep II- and GST-tagged scFvs in the eluent were 98.4% and 83.0%, respectively. Thus, both the short peptide Strep II and GST protein are suitable fusion tags for the affinity purification of proteins from silkworm larvae.     

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Efficient production of recombinant protein in Spodoptera frugiperda/AcNPV system utilizing silkworm hemolymph

Silkworm hemolymph when added at 5% (v/v) to medium increased the production of recombinant -galactosidase in Spodoptera frugipera/ Autographa californica nuclear polyhedrosis virus (AcNPV) system by 4.5-fold. Silkworm hemolymph also increased the host cell longevity by two-fold. Viability of the host cell was maintained at a constant level for 6 days after baculovirus infection in the medium containing 5% silkworm hemolymph, while host cells began to die 3 days after infection in the medium without hemolymph.     

Development of an Oligidic Diet forin VitroRearing ofTrichogramma galloiZucchi andTrichogramma pretiosumRiley

The goal of this research was to develop an artificial diet for rearingTrichogramma galloiandTrichogramma pretiosum,which are important egg parasitoids of several pests in Brazil. The diet was based on different proportions of hemolymph ofHelicoverpa zealarvae, chicken egg yolk, powdered milk solutions, bovine fetal serum, and an extract fromHeliothis virescenseggs. The ideal volume of diet for rearingT. galloiwas also evaluated. The parasitoids showed different nutritional requirements for their development. The best proportion of diet components forT. galloiwas 70% hemolymph, 20% egg yolk, 10% fetal serum, and 0.2% streptomycin, and forT. pretiosumit was 70% hemolymph, 20% egg yolk, 5% fetal serum, 5% egg extract, and 0.2% streptomycin. Egg yolk was found to be essential for the development of both parasitoids, and the powdered milk solution was not an important component in the diet. Development was not obtained in diets with hemolymph concentrations lower than 40%. A 3- to 4-μl amount of diet was the ideal volume on which to rearT. galloiwith the techniques reported.     

Kinetic effect of silkworm hemolymph on the delayed host cell death in an insect cell-baculovirus system

The kinetic effect of silkworm hemolymph on host cell viability during a baculovirus-induced insect cell death process was investigated. Host cell viability after viral infection is important for replication of the baculovirus DNA containing a recombinant gene and expression of the cloned gene. The baculovirus-induced insect cell death process can be divided into a delay phase and a first-order death phase, which are characterized by a delay time (t(d)) and a specific death rate (k(d)), respectively. For 0-10% silkworm hemolymph in the media, higher concentrations resulted in longer delay times and lower specific death rates. By adding 10% silkworm hemolymph, the delay time increased from 72 to 164 h, and the specific death rate was reduced from 13.8 x 10(-)(3) to 6.0 x 10(-)(3) h(-)(1). In addition, host cell viability correlated with DNA fragmentation, which is the biochemical hallmark of apoptosis. This indicates that the silkworm hemolymph inhibits the baculovirus-induced insect cell apoptosis. However, the silkworm hemolymph did not affect the number of hypothetical targets, which represents host cell susceptibility to the baculovirus. The concentration of fetal bovine serum (FBS) in the medium did not affect the delay time, while lower concentrations of silkworm hemolymph resulted in shorter delay times. This means that the substance which increases the longevity of the host cell is not in the FBS but in the silkworm hemolymph.     

Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.     

Improved secretion of molecular chaperone‐assisted human IgG in silkworm,and no alterations in their N‐linked glycan structures

Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 μg/larva and 78.0 μg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 μg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N‐linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manα1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N‐linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N‐glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post‐translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N‐acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

Production of a Cellulase in Silkworm Larvae using a Recombinant Bombyx mori nucleopolyhedrovirus lacking the Virus-encoded Chitinase and Cathepsin Genes

The expression efficiency of an insect-derived cellulase was assayed in silkworm larvae infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) mutants lacking the virus-encoded chitinase (chiA) and/or cathepsin (v-cath) genes. Expression was increased by approx. 10% in mutants lacking chiA or v-cath and 17% in a mutant lacking both chiA and v-cath compared with that of the unmodified recombinant BmNPV. The recombinant BmNPV lacking both chiA and v-cath can therefore be used for a large-scale production of foreign proteins in silkworms. Revisions requested 27 September 2005 and 16 December 2005; Revisions received 6 December 2005 and 3 February 2006     

Enhancing effect of a protein from <Emphasis Type="Italic">Lonomia obliqua</Emphasis> hemolymph on recombinant protein production

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.     

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of <Emphasis Type="Italic">Lonomia obliqua</Emphasis>

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system.     

YptV2p,a membrane-associated small G protein abundant during embryogenesis in the green algaVolvox carteri

Summary The multicellular fresh-water algaVolvox carteri contains at least six small G protein-encoding genes (ypt genes) whose products are probably involved in intracellular vesicle transport. Four of them, YptV1, YptV3, YptV4, and YptV5, have been isolated and characterized previously. Here we report the cloning ofyptV2 cDNA, the production of recombinant His-tagged YptV2p protein (reYptV2p) inE. coli, and the analysis of its GTPase activity and intracellular localization. YptV2p is predominantly present in dividingVolvox embryos. It is a membrane-associated protein which is localized to the cell periphery (plasma membrane or plasma-membrane-associated vesicles), probably by a lipid moiety. Purified,E. coli-expressed YptV2p binds GTP specifically, and has a typically low intrinsic GTPase activity (k_cat=0.004/min), which is enhanced by a GTPase activating protein activity present inVolvox. Our observations suggest a role of YptV2p in secretion, with a peak during the rapid cleavages of theVolvox embryo.Abbreviations GAP GTPase-activating protein - re recombinant     

Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm’s hemolymph due to both the resistant characters of spores and the addition of enterokinase site.