A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus

The expression characteristics of the pl0, polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and fl-galactosidase. In these systems, JHE is exported from the cell and fl-galactosidase is localized to the cytosol. Expression of JHE from the basic, pl0 and polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of fl-galactosidase and JHE under control of the pl0 promoter are higher relative to expression under control of the polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.

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“…lacZ (Possee & Howard, 1987) and AcMP1. lacZ (Hill-Perkins & Possee, 1990) was compared with expression of JHE by AcUW2(B). JHE, AcRP23.…”

Section: Methodsmentioning

confidence: 99%

“…The 6.9K arginine-rich basic protein is expressed during the late phase of viral infection. The basic protein promoter has been duplicated in place of the polyhedrin gene in the plasmid pAcMP1 (Hill-Perkins & Possee, 1990).…”

Section: Introductionmentioning

confidence: 99%

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Bonning

Roelvink

Vlak

et al. 1994

Journal of General Virology

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“…AcMLF9.ScathL expresses a functional flesh fly cathepsin L protease (ScathL) (Harrison and Bonning, 2001), AcMLF9.ScathL.C146A expresses a catalytic site mutant of ScathL , and the expression of both proteins was directed by the AcMNPV p6.9 promoter (Harrison and Bonning, 2000;Hill-Perkins and Possee, 1990). Budded virus stocks were produced in Sf21 cells.…”

Section: Insect Cells Insects and Virusesmentioning

confidence: 99%

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

Tang

Sivakumar

et al. 2008

Journal of Insect Physiology

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ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD 50 ) for purified ScathL injected into fifth-instar H. virescens was 11.0 μg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisumwith a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 μg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system. RightsWorks produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted. AbstractScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD 50 ) for purified ScathL injected into fifth-instar H. virescens was 11.0 mg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 mg/larva did not produce any effect i...

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“…The expression of both proteins was directed by the AcMNPV p6.9 promoter (Harrison and Bonning, 2000b;Hill-Perkins and Possee, 1990), additional recombinant viruses AcMLF9.ScathL.hsp70/LacZ and AcMLF9.ScathL.C146A.hsp70/LacZ were constructed to express ScathL or ScathL.C146A…”

Section: Insect Cells Insects and Virusesmentioning

confidence: 99%

“…The expression of both proteins was directed by the AcMNPV p6.9 promoter (Harrison and Bonning, 2001;Hill-Perkins and Possee, 1990) along with the vankyrin gene or epidermal growth factor gene 1.0 under control of the constitutively expressed hsp70 promoter . Polyhedra will be generated and purified as described previously (Harrison and Bonning, 2001).…”

Section: Viruses and Insectsmentioning

confidence: 99%

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

Tang

Lei

et al. 2007

Tissue and Cell

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A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus

Cited by 84 publications

References 19 publications

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

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