Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A



Langari J¹ ; Golkar M² ; Khanahmad H³ ; Karimipoor M^{4, 5} ; Arezumand R^{4, 5} ; Behzadi R⁶ ; Ahangaricohan R⁷ Show Affiliations
Source: Journal of Isfahan Medical School Published:2014

Abstract

Background: Nowadays, in many studies related to the treatment of various cancers, toxic compounds are targeted against cancer cells. One of the most effective compounds is Pseudomonas exotoxin A. The purpose of this study was to investigate the expression, purification, and in-vitro evaluation of a short form of the toxin in a prokaryotic expression system. Methods: The short form of the toxin (PE38) was amplified via polymerase chain reaction (PCR) using primers containing HindIII and NdeI restriction enzyme sites from pUC57-PE38. The polymerase chain reaction product was digested and subcloned in the pET-26b expression vector. The expression vector was separately transformed into the BL21(DE3), BL21(DE3) plys S and Rosetta Escherichia coli strains. Recombinant bacteria were cultured and induced and the resulted PE38 protein purified using metal affinity column chromatography. The toxicity effect of PE38 protein was assessed on HUVEC and 293KDR eukaryotic cells. Findings: The gene was successfully cloned into the expression vector and the accuracy of construct was confirmed via restriction map analysis and sequencing. The expression was found more in BL21(DE3) than the other strains. The toxicity effect was observed at the same level for both HUVEC and 293KDR cells. Conclusion: The lethal dose of truncated toxin is more than the previous study (1000-fold), where the targeted vascular endothelial growth factor (VEGF121) was fused to the truncated toxin. © 2014, Isfahan University of Medical Sciences(IUMS). All rights reserved.