2005 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, 2005 through December 31, 2006
Project Amount: $320,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , Sustainability
The overall goal of this research project is to develop a more cost-effective method for mass-producing baculovirus insecticides. The specific objective of this 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. First, modifying the base sequence of the fp25K gene (whose mutation leads to FP mutants). This research is being conducted in the Bonning Laboratory (Iowa State University). Second, expressing 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
Expression of FP25K Protein Under Baculovirus Late p6.9 Promoter in 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 native promoter under which the FP25K protein is expressed in A. californica is a late promoter (FP25K promoter). Hence, it is proposed to produce stably transformed insect cell lines (Sf-9 and Sf-21) that express the FP25K protein under the baculovirus late promoter (p6.9 promoter). It also is proposed to express the FP25K protein under Drosophila melanogaster hsp70 promoter in host Sf-9 cells.
Construction of the p6.9-fp25k-pIB Vector. We already have inserted the p6.9:fp25k gene cassette in the pIB vector (Invitrogen), which can be stably integrated into the Sf-9 genome. For this process, we conducted the polymerase chain reaction (PCR) amplification of the p6.9 promoter region from AcMLF9 (from Bonning’s Laboratory) and fp25k gene from the puc19-fp25k vector (Feiss Laboratory), followed by another PCR amplification of the combined cassette of p6.9-fp25k. Then this p6.9-fp25k cassette is integrated in pIB vector at the HinDII and EcoRI restriction site of the multiple cloning sites of the plasmid. This vector can be used to transfect the host insect cells instigating them to express FP25K protein.
Also, we currently are inserting the hsp70:fp25k gene cassette in the pIB vector to replicate the same process.
Evidence of Cellular FP25K Protein Expression. After transfection of the Sf-9 cells with the p6.9-fp25k-pIB or hsp70-fp25k-pIB vector DNA, we need to analyze the whole cell lysates by sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and Western Blot to determine if the FP25K cellular expression could be detected. If we cannot see a clear difference between the wild type (WT) Sf-9 cell and the Sf-9 cell having fp25K gene in SDS PAGE gel, then we need to perform the Western Blot to check the expression of FP25K. For this we need the FP25K antiserum.
Production of FP25K Antiserum From the FP25K Protein Expressed in Escherichia coli. We have inserted the fp25K gene in pMAL-c2x vector (New England Biolab), which provides a method for expressing and purifying a protein produced from the cloned gene. The fp25k gene is inserted downstream from malE gene of E. coli, which encodes maltose binding protein (MBP). This pMAL-c2x vector has a strong tac promoter to give a high-level expression of FP25K, coupled with the MBP, resulting in a MBP fusion protein. We can purify this MBP fusion protein using MBP’s affinity to maltose. As the pMAL-c2x vector encodes the site for enterokinase (a specific protease), the MBP can be cleaved from FP25K, our protein of interest. The FP25K protein purified this way can be used for the production of FP25K anti-serum.
Production of Virus. Two separate control 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. The BacPAK is a polyhedrin negative virus that has a unique Bsu36 I linearizable site. All these viruses, along with the WT AcMNPV, have been plaque purified three times and are ready to use.
We will transfect the Sf-9 and Sf-21 cells with the p6.9-fp25k-pIB DNA to construct the recombinant Sf-9 cells and check by Western Blot if they express the FP25K protein under control of the p6.9 promoter. It is expected 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 will test AcMNPV production in these cells in a continuous bioreactor system consisting of agitated bioreactors in a series. Furthermore, AcMNPV modified in the Bonning Laboratory will be tested in this continuous system.