Assay of the Week: Disabled Insecticidal Protein (DIP) assay

Assay of the Week: Disabled Insecticidal Protein (DIP) assay

Application: Screening for the alternative MoA (different insect receptors binding) of insecticidal pore-forming toxins.

Field: AgBiotech, entomology, protein science.

Background: Cry proteins, also known as crystal proteins, are a class of insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). These proteins/toxins are notable for their insecticidal properties and are widely used in biotechnology and agriculture as a natural means of pest control. After being ingested by susceptible insects, Cry proteins are activated in the insect’s gut. They bind to specific receptors in the gut lining, forming pores that disrupt the gut membrane, leading to cell lysis and ultimately causing the death of the insect.

There are various Cry protein variants (such as Cry1, Cry2, Cry3, etc.), each with specific toxicity against particular insect species. Different Cry proteins have differing levels of specificity, targeting different pests. Genes encoding Cry proteins have been incorporated into crops through genetic engineering. Genetically modified crops, known as Bt crops (such as Bt corn and Bt cotton), express Cry proteins, providing the plants with inherent resistance against specific insect pests. However, the insect population might develop resistance against a particular Cry toxin due to genetic variability over many generations. As a result, there is a continuous search for novel Cry proteins in metagenomics databases or by altering the activity of existing Cry proteins using genetic engineering techniques.

Assay detail: DIP assay was introduced by Jerga et al. from Bayer Corp Science in 2019 (https://pubmed.ncbi.nlm.nih.gov/30605769/ ) to enable screening for Cry proteins that bind to different insect receptors. First, a disabled insecticidal Cry protein is generated using the directed mutation method. This is a protein variation that sustains receptor binding function but misses the pore-forming activity (hence the insecticidal activity, known as DIP). In the assay, excess amounts of DIP and Cry protein/toxin of interest are incubated with BBMV (for in vitro testing) or insect (for in vivo testing). If DIP and toxin of interest bind to different receptors (different MoA), there will be a signal (insect size or readout). Since this is a competitive assay, one can then use a concentration-dependent response formula to determine LC50 for the Cry protein. Find more details in the references below.

What I think about this assay: insects’ gut biology is very complex! Except for a few, the specific receptors for Cry proteins are unknown. DIP assay offers a method to screen for novel toxin/receptor binding using a panel of Cry-proteins with known receptor binding. Once a toxin is selected, it can be further engineered and added to the DIP panel for future screening. I personally like the interpretability of the DIP assay and the fact that it can be performed in HT (in the case of in vitro testing) for library screening. These make DIP an excellent assay for the early ranking of toxins in the discovery phase. Candidates from this phase then can enter the secondary screening pipeline