Cloning of habutobin cDNA and antithrombotic activity of recombinant protein

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Habutobin is a protein found in Trimeresurus flavoviridis (habu snake) venom.
• Understanding its structure-function relationship is crucial for potential therapeutic applications.

Purpose of the Study:

• To clone and express habutobin.
• To investigate the functional properties of recombinant habutobin, particularly its effects on blood coagulation and platelet aggregation.
• To assess the utility of different expression systems for producing functional habutobin.

Main Methods:

• Cloning of habutobin cDNA from Trimeresurus flavoviridis.
• Expression of recombinant habutobin using bacterial (pET-r-habutobin) and baculoviral (AcNPV-r-habutobin) systems.
• Purification and refolding of recombinant proteins.
• Assays for fibrin forming activity and collagen-induced platelet aggregation.

Main Results:

• Habutobin cDNA was successfully cloned and translated into a 236-amino acid protein (25.7 kDa).
• Recombinant habutobin was produced and purified via bacterial and baculoviral systems.
• AcNPV-r-habutobin dose-dependently enhanced fibrin formation and inhibited platelet aggregation.
• Refolded pET-r-habutobin showed multiple bands, suggesting potential protein processing.

Conclusions:

• The baculoviral expression system is highly effective for producing functional recombinant habutobin.
• AcNPV-r-habutobin exhibits significant procoagulant and antiplatelet activities.
• Further studies on the structure-function relationship of habutobin are warranted for developing novel antithrombotic drugs.