Expression of functional ricin B chain using the baculovirus system

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Ricin B chain (RTB) is crucial for ricin's cellular entry and toxicity.
• Efficient expression systems are needed for recombinant RTB production.
• Understanding RTB's biological activity is key for therapeutic applications.

Purpose of the Study:

• To establish a baculovirus expression system for producing functional recombinant ricin B chain (RTB).
• To characterize the expressed RTB, including its glycosylation, molecular mass, and cellular localization.
• To evaluate the biological activity and potential applications of the recombinant RTB.

Main Methods:

• Insertion of the RTB coding sequence into a baculovirus transfer vector.
• Transfection of Spodoptera frugiperda Sf9 cells and selection of recombinant viruses.
• Immunoblotting and immunofluorescence for RTB detection and localization.
• Purification using immobilized lactose and assessment of biological activity.

Main Results:

• Optimal RTB expression achieved 72 hours post-infection with a multiplicity of infection of 3.
• Recombinant RTB was glycosylated, with an apparent molecular mass of 34 kDa, and located in the endoplasmic reticulum/Golgi.
• Secretion was enhanced by lactose; purification yielded >2 mg/l.
• Recombinant RTB demonstrated identical binding, internalization, and reassociation with ricin A chain compared to native RTB.

Conclusions:

• The baculovirus expression system effectively produces functional, glycosylated ricin B chain (RTB).
• Recombinant RTB retains native biological activities, including lectin binding and translocation.
• The N-terminal extension does not impair RTB translocation, supporting its use in delivering antigenic peptides for immunotherapy.