Document Type : Research Paper
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Background: Acetate accumulation in the culture medium is known as an inhibitor in recombinant protein production in Escherichia coli. Various approaches have been proposed and evaluated to overcome this challenge and reduce the concentration of acetate. In this study, we examined the effect of acetate kinase A antisense on acetate production rate in E. coli We also used PAMAM dendrimers as a suitable delivery agent for antisense transformation into E. coli host cell. Objective: This study aimed to decrease acetate production as a by-product using an antisense-dendrimer complex to increase mass cell and subsequently recombinant Albumin production in E. coli. Materials and Methods: Here, to study the effect of this treatment on recombinant protein production, we used pET22b/ HAS construct. The ackA gene expression was inhibited by designed antisense to reduce acetate concentration in culture medium. AckA antisense was transferred to E. coli by PAMAM dendrimer. Finally, ackA expression and recombinant Albumin production were evaluated Real-Time PCR and densitometry, respectively. Results: Our data showed, designed antisense lead to reduction of acetate kinase gene expression and subsequently acetate concentration in the culture medium. Finally, acetate concentration reduction and cell mass increase result in enhanced recombinant Alb production in the treated group (1.25 mg.mL-1) compare to the control group ( 0.59 mg.mL-1). Conclusions: Reduction of acetate in E. coli fermentation process decreased the recombinant Alb production following cell growth and cell mass increase. In the current study, we showed that an antisense can be a useful tool for ackA gene expression reduction. Also, we noted that PAMAM dendrimer could be a proper delivery agent for oligonucleotide antisense transformation into bacterial cells.