Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli.

Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli. Cell Mol Biol (Noisy-le-grand). 2018 Jan 31;64(1):64-69 Authors: Qaiser H, Aslam F, Iftikhar S, Farooq A Abstract Pseudomonas aeruginosa exotoxin A (PE) is a bacterial toxin composed of three domains namely: cell binding, translocation and enzymatic domain. The cytotoxic activity of PE is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of EF-2. PE can be genetically modified to fight cancer. In this regard, a truncated and modified form of PE was produced that could be used for more potent immunotoxins. This modified form termed PE38KDEL was completely devoid of cell binding domain and parts of translocation domain II and Ib which are reported to be inessential for cytotoxicity of the toxin. The resultant expressed protein consisted of the essential translocation domain II and catalytic subunit (domain Ib, III). The deletions in the exotoxin A gene for truncated protein production were made via overlapping PCR extension. The amplicon was cloned in pTZ57r-T vector for DNA works and sub cloned in pET22b expression vector.  It is demonstrated here that PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expression was optimized at 0.5 mM IPTG...
Source: Cellular and Molecular Biology - Category: Molecular Biology Tags: Cell Mol Biol (Noisy-le-grand) Source Type: research