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[Cloning and expression of Tachypleus tridentatus factor C]
Dong-Ning Wang1, Jie-Wu Liu, Lin Chen
1College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai 200030, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao Acta Biochimica Et Biophysica Sinica
|April 18, 2002
Summary
Researchers successfully cloned and expressed active Factor C, an endotoxin-sensitive enzyme crucial for horseshoe crab coagulation. This advancement holds potential for developing new anti-lipopolysaccharide (LPS) treatments.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Context:
- Factor C (FC) is a key enzyme in the horseshoe crab hemolymph coagulation cascade.
- Its lipopolysaccharide (LPS)-binding capability suggests therapeutic potential for anti-LPS treatments.
- Understanding FC's structure and function is vital for harnessing its commercial value.
Purpose:
- To clone and express recombinant Factor C (FC) from Chinese Tachypleus tridentatus.
- To confirm the activity of the refolded recombinant FC.
- To investigate the potential for self-cleavage of recombinant FC.
Summary:
- Primers were designed based on known Japanese horseshoe crab FC sequences.
- Total RNA from Chinese Tachypleus tridentatus amebocytes was used for cDNA cloning via RT-PCR.
- The FC cDNA was cloned into a pET-28a (+) vector, creating the pET-FC plasmid, and expressed in E. coli BL21 (DE3).
- Recombinant FC was expressed as inclusion bodies and subsequently refolded, showing in vitro activity via bacteriostatic assay.
- Western blot analysis indicated potential partial self-cleavage of the recombinant FC.
Impact:
- Successful expression and refolding of active recombinant Factor C.
- Provides a foundation for further studies on FC's enzymatic properties and therapeutic applications.
- Demonstrates the feasibility of producing functional horseshoe crab Factor C for research and potential drug development.