Expression And Activity Identification Of Truncated Pseudomonas Exotoxin A In Escherichia Coli

Objective:Pseudomonas Exotoxin A (PE) is one of the pathogenic proteins that is secreted by Pseudomonas aeuriginosa. It helps the bacteria invade body tissues. PE is composed of a single peptide chain, and inhibits protein synthesis in eukaryotic cells and causes cell death. Since 1980s, several plant toxins and bacterial toxins have been coupled to antibodies and cytokines to make immunotoxins specific for surface properties in order to kill target cells specifically. These immunotoxins are now being applied for treatment of cancers, autoimmune diseases, and chronic infectious diseases, especially for the treatment of malignant tumor. At present, many researches of imminotoxins containing PE have reached the stage of clinical trials. Some trials of these immunotoxins targeting tumors are currently in progress. However, unexpected toxicities appears to produce intolerable side effects, for example, fever, chill, vascular leak syndrome (VSL), injury of liver or kidney.In this study, the recombinant plasmid pGEX-4T-1/PE38KDEL was constructed, and transformed into E. Coli BL21(DE3) by chemical transformation. The fusion protein GST-PE38KDEL was expressed in E. Coli after induced by IPTG, and purified by GSTrap FF column. The cytotoxicity of the fusion protein was detected by MTS assay in vitro. These results provided some useful data to mutate PE38KDEL residues to ameliorate non-specific toxic and construct more effective immunotoxins with modified PE38KDEL.Methods:(1) The construction of prokaryotic expression vector by using truncated Pseudomonas Exotoxin A (PE38KDEL): we acquired PE38KDEL gene sequence from GenBank. According to the sequence, the primers for amplifying PE38KDEL gene were designed by software Primer Primier 5.0. PE38KDEL gene was amplified by PCR from the plasmid pRKL/IL18-PE38KDEL. PCR products were cloned into prokaryote expression vector pGEX-4T-l after being digested by EcoRⅠand NotⅠto construct the recombinant plasmid pGEX-4T-1/PE38KDEL. The recombinant plasmid was identified by restriction endonucleases digestion, PCR and sequence analysis.(2) The expression and characteristics of fusion protein PE38KDEL with GST in E. colt After the pGEX-4T-1/PE38KDEL transformed into E. coli BL21(DE3) and induced by IPTG, the expression product was obtained. The molecular weight and specificity of the fusion protien were identified by SDS-PAGE and Western blot. The fusion proteins GST-PE38KDEL with GST was purified through the GSTrap FF column. The cytotoxicity of fusion protein GST-PE38KDEL was checked by MTS assay in vitro.Results(1) After verification of the cloning fidelity by restriction endonucleases digestion, PCR, and sequencing, the successful constructions of prokaryote expression plasmids pGEX-4T-l/PE38KDEL were confirmed. The sequence of PE38KDEL was identical to that of PE in GenBank and the ORF was also not changed.(2) The BL21(DE3) transformed with pGEX-4T-I /PE38KDEL expressed fusion protein GST-PE38KDEL. The molecular weight of expression product was identical to the expected size, and showed the fusion protein with Mr of 63 000. The expression product was able to react with the specific anti-PE rabbit sera when it was detected by Western blot. The soluble fusion protein was purified by GSTrap FF column. The protein of Mr 63 000 was obtained when the GSTrap FF column was washed with 10 mmol/L Elution buffer. The cytotoxicity of fusion protein was detected by MTS assay. The result indicated that it was none or slightly toxic in several cell lines by the addition of the fusion protein GST-PE38KDEL to a final concentration of 1～10 ug/ml, but the fusion protein GST-PE38KDEL had extremely cytotoxicity at 100～1000μg/ml.Conclusion(1) The prokaryote expression vectors coding truncated Pseudomonas Exotoxin A (PE38KDEL) were constructed successfully.(2) After the recombinant plasmid pGEX-4T-l/PE38KDEL transformed into E. coli BL21(DE3), large amounts of fusion protein GST-PE38KDEL were purified. By cytotoxicity assay, The target protein was extremely toxic in several cell lines at high concentration. The results provided some useful data to mutate PE38KDEL residues to ameliorate non-specific toxic and construct more effective immunotoxins with modified PE38KDEL.