Vida Mirzaie,1 Touba Eslaminejad,2 Homayoon Babaei,3 Seyed Noureddin Nematollahi-Mahani4,5

1Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 2Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran; 4Neuroscience Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; 5Afzal Research Institute (NGO), Kerman University of Medical Sciences, Kerman, Iran

Correspondence: Seyed Noureddin Nematollahi-Mahani Tel +983 433257343Email nnematollahi@kmu.ac.ir

Purpose: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1 (EF-1) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion.Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellmans method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector.Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellmans method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity.Conclusion: The designed construction with CMV and EF-1 promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.

Keywords: dual-promoter, vector, recombinant protein, drug delivery, Ellmans method, HEK-293

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

Follow this link:

Enhancing the Butyrylcholinesterase Activity in HEK-293 Cell Line by D | DDDT - Dove Medical Press