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Updated: Aug 25, 2026
Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
Published on: April 20, 2017
Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in
Abstract:
The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.
Insights
Bacillus thuringiensis subsp. israelensis and morrisoni produce mosquitocidal proteins. A new expression system successfully produced the 72-kDa CryIVD protein, showing comparable toxicity to natural strains.
Area of Science:
- Microbiology
- Molecular Biology
- Insecticide Development
Background:
- Bacillus thuringiensis subspecies israelensis and morrisoni possess mosquitocidal properties due to protein inclusions.
- Mosquitocidal activity is linked to specific proteins with molecular masses of 27, 72, 128, and 135 kDa.
Purpose of the Study:
- To determine the toxicity of individual mosquitocidal proteins.
- To utilize the pHT3101 shuttle vector for expressing the cryIVD gene in a bacterial host.
Main Methods:
- The cryIVD gene from B. thuringiensis subsp. morrisoni was expressed in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki using the pHT3101 vector.
- Parasporal inclusions of the 72-kDa CryIVD protein were analyzed for size, shape, and toxicity.
- Bioassays were conducted against fourth instars of Culex quinquefasciatus to determine lethal concentrations.
Main Results:
- Parasporal inclusions of the 72-kDa CryIVD protein produced via the expression system were comparable to those from the parental strain.
- The inclusions exhibited bar shapes (500 x 300 x 150 nm) and were visible via phase-contrast microscopy.
- The 50% lethal concentration (LC50) was 64 ng/ml against Culex quinquefasciatus, similar to the 55 ng/ml LC50 for the 72-kDa inclusion from B. thuringiensis subsp. israelensis.
- Expression of the cryIVD gene in Escherichia coli was minimal and only detectable by immunoblot analysis.
Conclusions:
- The pHT3101-B. thuringiensis expression system effectively produces the CryIVD protein with properties similar to its natural host.
- This system is a viable tool for expressing other Bacillus thuringiensis proteins.
- It holds potential for reconstitution experiments involving inclusions from mosquitocidal subspecies.
