Expression and characterization of a kinase from Chilo iridescent virus

Despite an annual $40 billion investment, economic losses due to insects average $200 billion a year. Traditional use of chemical pesticides is detrimental to human health due to tainted food and contaminated ground water. Pesticide usage also leads to loss of beneficial insects, outbreaks of secondary pests, and increased insecticidal resistance. A more selective alternative to traditional pest control, biopesticides are naturally occurring substances including microorganisms that are used in the control of insect pests. Biopesticides are also pesticidal substances produced by plants containing added genetic material (plant-incorporated protectants or PIPs). As part of an integrative pest management (IPM) strategy, biopesticides reduce pesticide residue on food, as well as increases preservation and biodiversity in the ecosystem. Chilo iridescent virus (CIV) is a large virus with a double-stranded DNA genome. As the type species of the family Iridoviridae, it infects many insects, induces mortality in a few, and has no effects in mammalian tissue culture. Use of this biopesticide is limited by its sensitivity to environmental extremes. But the Bilimoria laboratory has discovered that CIV protein extract (CVPE) induces apoptosis and inhibits host protein synthesis in insect tissue culture without viral gene expression. Research began into determining which component(s) of CVPE induce these effects. Primer walking of CIV genome identified an open reading frame (ORF) similar to B1R kinase of vaccinia virus. Identical to CIV ORF 389L, this 1236 bp gene has been designated iridovirus serine/threonine kinase (istk). My hypothesis is that ISTK can be expressed in a bacterial system and this recombinant protein will have kinase and insecticidal activity. Cloned in frame with either an N-Terminal or C-Terminal 6x Histidine purification Tag, the bacterial expressed, viral recombinant enzyme showed low but statistically significant kinase activity versus controls which includes buffer, BSA, mock preparations and ISTK inhibited with the kinase inhibitor staurosprorine. However bioassays against green peach aphids only demonstrated significant statistically difference in mortality between cotton leaf discs treated with ISTK versus untreated, buffer control and BSA negative controls. One-Way ANOVA and Tukey’s Range test determined that there was no statistical difference between the ISTK treatment and mock, heat-inactivated mock and heat-inactivated ISTK controls. However the potential of ISTK as a biopesticide or a plant-incorporated protectant (PIP) is still inconclusive. The Pichia expressed ISTK subfragment, iridoptin appears to have higher kinase activity versus its bacterial form and showed significant insecticidal activity. This suggests that post-translational modification may influence in-vitro kinase activity and insecticidal capabilities of this viral protein. This research involved finding an alternative expression system to Pichia in order to synthesize a recombinant form of ISTK that did not fragment into iridoptin. Determining kinase activity and post-translational modifications of the original viral protein (from the virus) is required for the selection of any expression system for this viral enzyme, and the resulting recombinant protein.