Pseudomonas aeruginosa
(PA) is a leading cause of nosocomial infections and death in cystic
fibrosis patients. The study was conducted to evaluate the
physicochemical structure, biological activity and serum stability of a
recombinant anti-PcrV single chain variable antibody fragment
genetically attached to the mCH3cc domain. The stereochemical properties
of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular
interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli
BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological
activity of the proteins in inhibition of PA induced hemolysis as well
as cellular cytotoxicity was assessed. In silico analysis revealed the
satisfactory stereochemical quality of the models as well as common
residues in their interface with PcrV. The structural differences of
proteins through circular dichroism spectroscopy were confirmed by NMR
analysis. Both proteins indicated inhibition of ExoU positive PA strains
in hemolysis of red blood cells compared to ExoU negative strains as
well as cytotoxicity effect on lung epithelial cells. The ELISA test
showed the longer serum stability of the YFL001 molecule than YFL002.
The results were encouraging to further evaluation of these two scFv
molecules in animal models.