Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A
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...
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| Format: | Article |
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Isfahan University of Medical Sciences
2014-09-01
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| Series: | مجله دانشکده پزشکی اصفهان |
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| Online Access: | http://jims.mui.ac.ir/index.php/jims/article/view/3199 |
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| author | Jahangir Langari Majid Golkar Hossein Khanahmad Morteza Karimipoor Roghayeh Arezumand Ramazan Behzadi Reza Ahangari-Cohan |
| author_facet | Jahangir Langari Majid Golkar Hossein Khanahmad Morteza Karimipoor Roghayeh Arezumand Ramazan Behzadi Reza Ahangari-Cohan |
| author_sort | Jahangir Langari |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T19:28:28Z |
| format | Article |
| id | doaj.art-4ab5ba2a92b34ac1a9355cfd8e540dc4 |
| institution | Directory Open Access Journal |
| issn | 1027-7595 1735-854X |
| language | fas |
| last_indexed | 2024-03-12T19:28:28Z |
| publishDate | 2014-09-01 |
| publisher | Isfahan University of Medical Sciences |
| record_format | Article |
| series | مجله دانشکده پزشکی اصفهان |
| spelling | doaj.art-4ab5ba2a92b34ac1a9355cfd8e540dc42023-08-02T04:43:04ZfasIsfahan University of Medical Sciencesمجله دانشکده پزشکی اصفهان1027-75951735-854X2014-09-0132294111011181655Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin AJahangir Langari0Majid Golkar1Hossein Khanahmad2Morteza Karimipoor3Roghayeh Arezumand4Ramazan Behzadi5Reza Ahangari-Cohan6PhD Candidate, Department of Molecular Medicine, Biotechnology Research Centre, Pasteur Institute of Iran, Tehran, IranAssistant Professor, Department of Parasitology, Pasteur Institute of Iran, Tehran, IranAssistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IranAssistant Professor, Department of Molecular Medicine, Pasteur Institute of Iran, Amol, IranPhD Candidate, Department of Molecular Medicine, Pasteur Institute of Iran, Amol, IranPasteur Institute of Iran, North Research Center, Amol, IranAssistant Professor, Department of Rabies, Pasteur Institute of Iran, Tehran, IranBackground: 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.http://jims.mui.ac.ir/index.php/jims/article/view/3199Pseudomonas auroginosa exotoxin APE38CloningExpression |
| spellingShingle | Jahangir Langari Majid Golkar Hossein Khanahmad Morteza Karimipoor Roghayeh Arezumand Ramazan Behzadi Reza Ahangari-Cohan Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A مجله دانشکده پزشکی اصفهان Pseudomonas auroginosa exotoxin A PE38 Cloning Expression |
| title | Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A |
| title_full | Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A |
| title_fullStr | Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A |
| title_full_unstemmed | Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A |
| title_short | Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A |
| title_sort | cloning expression and evaluation of pseudomonas aeruginosa exotoxin a |
| topic | Pseudomonas auroginosa exotoxin A PE38 Cloning Expression |
| url | http://jims.mui.ac.ir/index.php/jims/article/view/3199 |
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