2004 Progress Report: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)EPA Grant Number: R831421
Title: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)
Investigators: Murhammer, David W. , Bonning, Bryony C. , Feiss, Michael G.
Institution: University of Iowa , Iowa State University
EPA Project Officer: Richards, April
Project Period: January 1, 2004 through December 31, 2006 (Extended to December 31, 2008)
Project Period Covered by this Report: January 1, 2004 through December 31, 2005
Project Amount: $320,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , Sustainability
The long-term objective of this research project is to develop a more cost-effective method for mass producing baculovirus insecticides. The specific objective of the research project is to develop methods to overcome the accumulation of few polyhedra (FP) mutants that would otherwise occur upon repeated baculovirus passage in cell culture. This includes two approaches. The first is modifying the base sequence of the fp25k gene (the mutation of which leads to FP mutants). This research is being conducted in the Bonning laboratory (Iowa State University). The second approach is to express the FP25K protein from the host cell genome. This research is being conducted in the Murhammer laboratory (University of Iowa).
FP25K Protein Expression in Sf-9 Host Cells
IE1 and IE1-FP25K Stability Incorporated Into Sf-9 Cells. The FP25K protein becomes mutated when Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is serially passaged in Sf-9 cells. The nature of this mutation most often takes the form of host cell DNA insertions. Because the host cell is not mutated during infection, it was proposed as a stable site for FP25K expression. The baculovirus immediate early promoter (IE1) and IE1:fp25k gene cassette has been stably integrated into the Sf-9 genome.
Sequence Evidence of IE1 and IE1-FP25K Incorporation Into the Sf-9 Genome. DNA from the Sf-9, IE1, and IE1-FP25K was isolated (DNAzol, Invitrogen) and sequenced to confirm that the IE1 and IE1:fp25k were inserted. The original fp25k isolation had a mutation at amino acid (aa) 92 changing an ATT to an ATA. This mutation is within the codon usage for the AcMNPV and did not change the resulting amino acid (Ile). Upon sequencing, the IE1-FP25K showed three changes from the original fp25k gene isolation. The first mutation at aa92 changed the original mutation from ATA back to the wildtype ATT. The second mutation occurred at aa101 and changed an Arg to Ser. The last mutation at aa138 changed the amino acid codon from Leu CTG to Leu CTA (this codon change is within the usage for AcMNPV). It is unknown at this time if these changes affect the activity of the resulting FP25K protein.
Evidence of Cellular FP25K Protein Expression. Whole cell lysates were analyzed by SDS Polyacrylomide Gel Electrophoresis and Western blot to determine if the IE1-FP25K cellular expression could be detected. Unfortunately, a clear difference was not seen between the different cell species either without or with infection (shown to increase the expression from the IE1 promoter).
Activity of Cellular FP25K During Infection With AcMNPV. Sf-9, IE1, and IE1-FP25K cells were infected with AcMNPV at multiplicity of infections (MOI) of 0.1, 1, and 10. The IE1-FP25K cells were able to produce polyhedra at an MOI of 0.1 at a level comparable to Sf-9 cells infected at an MOI of 10. The polyhedra were analyzed by transmission electron microscopy and found to contain significantly less virus than Sf-9:MOI 10 condition. Expression analysis for MOI 0.1 indicates that the IE1-FP25K can induce polyhedrin and FP25K mRNA approximately 10 hours earlier than in the Sf-9 or IE1 cells.
Activity of Cellular FP25K During Infection With AcFPβGal. Sf-9, IE1, and IE1-FP25K cells were infected at MOIs of 0.001, 0.01, and 0.1 with AcFPβGal. The AcFPβGal does not produce an intact FP25K protein. The IE1 and Sf-9 cells were able to produce low levels of polyhedra at an MOI of 0.1. The infected IE1-FP25K cells were able to produce polyhedra at all levels, and as the virus MOI increased so did the number of polyhedra. At MOI 0.01, the percent of cells containing polyhedra in the IE1-FP25K cells was equal to Sf-9 cells infected at an MOI of 10 with AcMNPV. When the polyhedra were analyzed by transmission electron microscopy, they contained very few viruses. Expression analysis for MOI 0.1 indicate that the IE1-FP25K can induce polyhedrin and FP25K:β-galactosidase mRNA approximately 10 hours earlier than in the Sf-9 or IE1 cells.
FP25K Protein Expression in AcMNPV E2 Virus
p10-FP25K, pIE1-FP25K and p6.9-FP25K. Strong baculovirus promoters may increase the level of FP25K produced, which may lead to the production of polyhedra-containing viruses. The fp25k gene was successfully incorporated into p10, pIE1, and p6.9 vectors.
Production of Virus. Initially, it was proposed to incorporate the p10-FP25K, pIE1-FP25K, and p6.9-FP25K into a virus that did not produce the FP25K protein. Two separate parent viruses were available, AcFPβGal and the ΔFP25K. The AcFPβGal produces a truncated FP25K and intact β-D-galactosidase protein, and the ΔFP25K has had the fp25K gene completely removed. Production of recombinant viruses is facilitated by linearization. To introduce a unique linearizable site in the parent virus, the pVL1393 vector was manipulated to contain a unique site (SbfI). The parent virus was then mixed with the pVL1393-SbfI vector, resulting in a virus that would be polyhedra negative and either blue or clear, respectively. Unfortunately, the double knockout viruses, not containing a functional fp25k or polyhedrin gene, have not expanded to date.
The BacPAK is a polyhedrin-negative virus that has a unique Bsu36 I linearizable site. It has been plaque-purified three times and is ready to use as the parent virus. This virus will be linearized and the plasmids containing the fp25k gene will be added. The resulting recombinant viruses will have a polyhedra positive phenotype.
FP25K Antisera Production
Antisera from Baculovirus FP25K Protein. A p10-FP25K virus was used to infect Sf-9 cells to produce FP25K protein for the production of anti-sera. The FP25K protein was purified using standard anion exchange chromatography. The protein was injected into a sheep and the resulting antisera was tested against Escherichia coli-produced FP25K (gift from Dr. Summers, Texas A&M). The antibody interaction was compared to antisera produced to FP25K made in E. coli with a factor Xa protease cleavage site and histidine tag (Dr. Summers). The E. coli-produced antibody interacted weakly with the baculovirus FP25K fraction, did not interact with the intact FP25K:factor Xa:histidine, and only weakly with the cleaved species. The E. coli antibody did interact with another fraction from the p10-FP25K anion exchange chromatography.
Increasing Antisera Specificity. In an attempt to enrich the antisera, a large volume of the E. coli factor Xa FP25K was produced and purified on a metal chelation column to homogeneity. The fraction was then treated with factor Xa to remove the histidine tag; unfortunately, this digestion step also digested 80 to 90 percent of the FP25K protein. This loss is most likely the result of the nonspecific nature of the factor Xa cleavage. The factor Xa cleaves at basic residues (lysine and arginine), which the FP25K protein has in abundance with a theoretical isoelectric point of 9.2. Alternatively, the p10-FP25K purification could be further refined by subjecting the positive fraction to further anion exchange purification steps. Using either procedure for isolating FP25K protein, the protein would then be bound to CNBr-activated resin. The antisera would then be applied to the resin to enrich the specificity of the antibody.
We plan to construct recombinant Sf-9 cells expressing the fp25k gene under control of the p6.9 promoter. We expect that this gene will be silent in the host cell genome until the cell is infected with AcMNPV. This will express the FP25K protein at the proper time post-infection. We also intend to test the production of AcMNPV production in these cells in a continuous bioreactor system consisting of agitated bioreactors in series. Furthermore, AcMNPV modified in the Bonning laboratory will be tested in this continuous system.