[Cloning and expression of Tachypleus tridentatus factor C].
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 it a 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.
Wang
Dong-Ning
DN
College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai 200030, China.
Liu
Jie-Wu
JW
Chen
Lin
L
Wang
Lie
L
Yang
Guan-Zhen
GZ
Wu
Xiang-Fu
XF
Zhang
Wei-Jie
WJ
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004792:Enzyme Precursors / Q000235:genetics* / Q000494:pharmacology; D015870:Gene Expression; D006737:Horseshoe Crabs / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics* / Q000494:pharmacology
D060829:Arthropod Proteins; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C