ABSTRACT
Background: Targeted cancer therapies have played a great role in the treatment of malignant tumors, in the recent years. Among these therapies, targeted toxin therapies such as immunotoxins, has improved the patient's survival rate by minimizing the adverse effect on normal tissues, whereas delivering a high dose of tumoricidal agent for eradicating the cancer tissue. Immunological proteins such as antibodies are conjugated to plant toxins or bacterial toxins such as Diphtheria toxin [DT] and Pseudomonas exotoxin A [PE] . In this case optimizing and expressing Diphtheria toxin and Pseudomonas exotoxin A which their binding domains are eliminated play a crucial role in producing the desired immunotoxins
Materials and Methods: We expressed the truncated DT and PE toxin in a genetically modified E.coli strain BL21 [DE3]. For this reason we eliminated the binding domain sequences of these toxins and expressed these proteins in an expression vector pET28a with the kanamycin resistant gene for selection. The optimization of Diphtheria toxin and Pseudomonas exotoxin A expression was due to different IPTG concentration, induction and sonication time
Results: We observed that the optimal protein expression of the Diphtheria toxin was gained in 4 hours of 0.4 mM IPTG concentration at 25celsius on the other hand the optimization of Pseudomonas exotoxin A protein occurred in 4 hours of 0.5 mM IPTG concentration at 25 celsius
Conclusion: Our study also showed lower IPTG concentrations could result in higher protein expression. By optimizing this procedure, we facilitate the protein production which could lead to acceleration of the drug development
ABSTRACT
Background: Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays [EIA]. Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy
Objectives: Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report
Materials and Methods: Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. coli BL21. Induction was performed by adding 1 mM isopropyl-beta-D-thiogalactopyranoside [IPTG] to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay [ELISA]
Results: The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting
Conclusions: The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactive HCV NS4B