EXPRESSION OF ACTIVE RECOMBINANT PALLIDIPIN, A NOVEL PLATELET-AGGREGATION INHIBITOR, IN THE PERIPLASM OF ESCHERICHIA-COLI

The platelet aggregation inhibitor pallidipin is a protein present in the saliva of the blood-sucking triatomine bug Triatoma pallidipennis. Expression of recombinant pallidipin in the periplasm of Escherichia coli was achieved by placing its coding sequence downstream of the alk aline phosphatase (APase) or trc promoter in frame with bacterial lead er peptide DNA sequences derived from APase or from the periplasmic fo rm of cyclophilin (Cph). In each case the DNA sequence of mature palli dipin was merged to the leader peptide coding part, either directly, o r while introducing additional amino acids, in order to assess their i nfluence on the activity of the leader peptidase and on the biological activity of the recombinant protein. All tested constructs gave rise to abundant periplasmic expression of pallidipin, which was then purif ied by a combination of cation- and anion-exchange chromatography foll owed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (similar to 19 kDa) and was correctly proc essed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencin g. The highest expression levels were obtained with the three APase-de rived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, c omparable with that of the native protein, except when an additional N -terminal lysyl-isoleucyl dipeptide was present, which resulted in an IC50 more than ten times higher.