Ancylostoma caninum anticoagulant peptide: cloning by PCR and expression of soluble, active protein in E. coli.
Ancylostoma caninum Anticoagulant Peptide (AcAP) is the major anticoagulant activity present in extracts of adult Ancylostoma caninum hookworms. This 8.7 kDa protein is a potent and specific inhibitor of human coagulation factor Xa. Using PCR, we have isolated a cDNA encoding for AcAP from an adult A. caninum cDNA library. The 5' end of the AcAP cDNA was identified by reverse transcription PCR (RT-PCR) using A. caninum cDNA and a 5' primer corresponding to a nematode spliced leader sequence. The AcAP cDNA was expressed in E. coli using a prokaryotic expression vector, and the recombinant fusion protein (rAcAP) was purified to homogeneity using nickel resin affinity chromatography and reverse phase HPLC. Purified rAcAP is comparable to the native protein in inhibitor activity, with an apparent equilibrium inhibitory dissociation constant (Ki*) for the inhibition of factor Xa of 265 +/- 71 pM. The purified protein also prolongs the prothrombin and partial thromboplastic times of human plasma in a dose dependent manner.
Cappello
M
M
Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520, USA.
Hawdon
J M
JM
Jones
B F
BF
Kennedy
W P
WP
Hotez
P J
PJ
D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D065427:Factor Xa Inhibitors; D015870:Gene Expression; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D011517:Prothrombin Time; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*
D065427:Factor Xa Inhibitors; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C094339:anticoagulant protein, Ancylostoma caninum