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Vol. 64 No. 1: Issue 1

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

https://doi.org/10.14715/cmb/2018.64.1.12
Hina Qaiser
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Farheen Aslam
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Saima Iftikhar
School of Biological Sciences, University of Punjab, Lahore, Pakistan
Adeen Farooq
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan

Corresponding Author(s) : Hina Qaiser

hinaqaiser@lgu.edu.pk

Cellular and Molecular Biology, Vol. 64 No. 1: Issue 1

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 concentration for induction as soon as the OD600 nm reached 0.6 with 6 hours of post induction culturing at 37°C.  The recombinant protein was expressed both as soluble and inclusion body forms however the expression of the soluble form was more pronounced.

Keywords

Pseudomonas aeruginosa exotoxin A Cytotoxic immunotoxin PE38KDEL Expression.
Qaiser, H., Aslam, F., Iftikhar, S., & Farooq, A. (2018). Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli. Cellular and Molecular Biology, 64(1), 64–69. https://doi.org/10.14715/cmb/2018.64.1.12
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Author Biographies

Hina Qaiser, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Lecturer, Department of Biology
Farheen Aslam, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Department of Biotechnology, Assistant Professor
Saima Iftikhar, School of Biological Sciences, University of Punjab, Lahore, Pakistan
School of Biological Sciences, Experimental Officer
Adeen Farooq, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Department of Biotechnology
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