Expression, purification and characterisation of recombinant pallidipin, a novel platelet aggregation inhibitor from the haematophageous triatomine bug Triatoma pallidipennis.
Pallidipin is a platelet aggregation inhibitor protein originating from the saliva of the haematophageous triatomine bug Triatoma pallidipennis. Its inhibitory effects are specific for collagen-induced platelet aggregation. The recombinant form of the protein was expressed in the periplasmic space of transformed Escherichia coli using a vector based on the alkaline phosphatase gene promoter and leader peptide. Recombinant pallidipin was purified in three chromatographic steps including cation exchange, anion exchange and size exclusion gel chromatography. SDS/PAGE and N-terminal amino acid sequencing showed that recombinant pallidipin had a molecular weight similar to that of the salivary protein (approximately 19 kDa) and had been correctly processed. The yield was 864 micrograms of pure protein from one litre of bacterial culture. The biological activity of recombinant pallidipin was assessed in a platelet aggregation assay using collagen at a concentration of 2 micrograms/ml as inducer, and the IC50 found to be 33 nM, similar to that determined for the native protein. When the collagen concentration used for induction was increased, higher pallidipin concentrations were also needed to achieve a comparable inhibition of platelet aggregation.
Haendler
B
B
Research Laboratories, Schering AG, Berlin, Germany.
Becker
A
A
Noeske-Jungblut
C
C
Krätzschmar
J
J
Donner
P
P
Schleuning
W D
WD
D000818:Animals; D003094:Collagen / Q000008:administration & dosage / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014224:Triatoma
D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C085906:pallidipin; D003094:Collagen