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 alkaline phosphatase (APase) or trc promoter in frame with bacterial leader peptide DNA sequences derived from APase or from the periplasmic form of cyclophilin (Cph). In each case the DNA sequence of mature pallidipin was merged to the leader peptide coding part, either directly, or while introducing additional amino acids, in order to assess their influence 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 purified by a combination of cation- and anion-exchange chromatography followed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (approximately 19 kDa) and was correctly processed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencing. The highest expression levels were obtained with the three APase-derived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, comparable 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.
Haendler
B
B
Research Laboratories of Schering AG, Berlin, Germany.
Becker
A
A
Noeske-Jungblut
C
C
Krätzschmar
J
J
Donner
P
P
Schleuning
W D
WD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000302:isolation & purification; D014224:Triatoma / Q000235:genetics*
D018076:DNA, Complementary; D010975:Platelet Aggregation Inhibitors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C085906:pallidipin