Cloning and
Expression of Tachypleus tridentatus Factor C
WANG Dong-Ning1, 2, LIU Jie-Wu2,
CHEN Lin, WANG Lie2, YANG Guan-Zhen2, WU Xiang-Fu2*,
ZHANG Wei-Jie1*
( 1College of Life Science and Technology, Shanghai Jiao
Tong University, Shanghai 200030, China; 2Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the
Chinese Academy of Sciences, Shanghai 200031, China )
Abstract Factor C is
an endotoxin-sensitive, intracellular serine protease zymogen which initiates
the coagulation cascade system in the horseshoe crab hemolymph. The special lipopolysaccharide
(LPS) binding activation of FC
makes ita potential drug for anti-LPS treatment and has a high
commercial value. Based on the sequence of reported FC from Japan horseshoe
crab, two pairs of primers were designed. The total RNA was extracted from
amebocytes of Chinese Tachypleus tridentatus and the cDNA was separated
into two parts and were cloned using RT-PCR, respectively. FCs from different geographical areas showed
high homology in sequence. The whole FC cDNA was cloned into pET-28a (+)
containing T7 promoter and recombinant expression plasmid pET-FC was
constructed. The recombinant plasmid was transformed into E. coli
BL21(DE3). Recombinant FC was expressed as inclusion body when the expression
strain was induced with 1 mmol/L IPTG. Refolded recombinant FC was confirmed to
be active by bacteriostatic assay in vitro. The results of Western blot
also suggested the recombinant FC may be able to cleave itself partly and
produced an extra immunoblot band.
Key words Tachypleus tridentatus factor C;
cloning; expression;
bacteriostatic activity
*Corresponding authors: ZHANG
Wei-Jie: Tel, 021-64955804; e-mail,
[email protected]; WU
Xiang-Fu: Tel, 021-64374430-5292;
Fax, 021-64338357; e-mail, [email protected]