Phage display of a biologically active Bacillus thuringiensis toxin

Area of Science:

• Molecular biology
• Biotechnology
• Insect toxicology

Background:

• Bacillus thuringiensis (Bt) insecticidal toxins often precipitate and inactivate when expressed in Escherichia coli.
• Limited molecular biology tools for Bacillus hinder genetic engineering of Bt toxins.
• Developing E. coli expression systems is crucial for advancing Bt toxin research and application.

Purpose of the Study:

• To develop a novel method for expressing active Bacillus thuringiensis (Bt) Cry1Ac toxin in E. coli.
• To overcome the challenge of insoluble Bt toxin precipitation in E. coli.
• To create a platform for genetic engineering and screening of Bt toxin variants.

Main Methods:

• Expressing activated Bt Cry1Ac toxin as a translational fusion with filamentous phage coat protein in E. coli.
• Utilizing phage particles to display the fusion protein.
• Assessing the viability, infectivity, and insecticidal activity of the displayed toxin.
• Employing Enzyme-linked immunosorbent assay (ELISA) and Western blot for antigenic analysis.
• Conducting micropanning with anti-Cry1Ac antibody to confirm toxin display.

Main Results:

• The fusion protein displayed on phage particles was viable, infectious, and retained insecticidal activity comparable to pure Cry1Ac toxin.
• Antigenic analysis confirmed the fusion protein's equivalence to native Cry1Ac toxin.
• Toxin-expressing phage particles were successfully identified using antibody-based panning.

Conclusions:

• Phage display provides a viable and advantageous system for expressing active Bacillus thuringiensis toxins in E. coli.
• This method facilitates genetic engineering and the construction of large toxin variant libraries for biopesticide development.
• Phage display overcomes limitations of previous E. coli expression systems for Bt toxins.