Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector.

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5'-triphosphates.

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm.

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.

Silkworm as a host of baculovirus expression.

While the Baculovirus Expression Vector System (BEVS) mainly uses insect cell lines, such as Sf9 cells, the robust high expression system using silkworm has also been developed. We have further improved technologies for enhancement of virus recombination, reduction of proteolytic degradation and aggregation, and more reliable promoters. These developments made it possible to achieve high and soluble expression of recombinant proteins. We review here such technology developments, advantage of using silkworm and some example applications. There are areas where this technology can be further improved as implicated in the end.

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.

Recombinant protein production by a Kaiko-baculovirus system.

Nucleopolyhedrovirus, a baculovirus, generates many intranuclear polyhedra in lepidopterous insects. The replacement of the polyhedra gene with a target gene, under a potent polyhedrin promoter, is widely used to express recombinant proteins. In this chapter, we describe the application of a highly efficient and reproducible baculovirus expression system with high throughput using Kaiko (silkworm).

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system.

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.

Establishment of a specific radioimmunoassay for bovine interferon tau.

A radioimmunoassay (RIA) was developed for quantification of bovine interferon (bIFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of bIFNtau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau.

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system.

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.

Establishment of a large-scale purification procedure for purified recombinant bovine interferon-tau produced by a silkworm-baculovirus gene expression system.

We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.

Method for purifying porcine mature interleukin-18 from silkworm haemolymph.

Recombinant porcine mature interleukin-18 (rPomIL-18) has been purified from the haemolymph of silkworms. After co-infection of two recombinant baculoviruses (BmAcpVL1392-IL-18-His and BmAcpVL1392-casp-1) into the silkworm, rPomIL-18 was produced and secreted into the haemolymph. Polyethylene glycol (PEG) 6000 was added to the haemolymph at 8% (v/w) to precipitate storage proteins which would otherwise bind non-specifically to the metal chelating column and the supernatant then was applied to Sepharose bonded with Ni2+. rPomIL-18 was eluted from the column using 100 mM imidazole buffer at pH 8 with a purity of 93.6%. Approximately 5.3 mg purified rPomIL-18 was obtained from 22 ml haemolymph. It could induce interferon-gamma formation from porcine peripheral blood mononuclear cells.

N-linked glycan structures of mouse interferon-beta produced by Bombyx mori larvae.

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.