Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus

VDAC2 protein identified to be over expressed in over-stunned chicken is a potential biomarker in differentiating stunned chicken and the voltage applied during the process. The VDAC2 gene was amplified by proofread DNA polymerase, then purified and inserted into pENTR-TEV-D-TOPO entry vector and...

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Main Authors:	Ahmad Hassan, Nurhidayah, Amid, Azura, Mohd. Salleh, Hamzah
Format:	Article
Language:	English
Published:	Journal of Pure and Applied Microbiology 2014
Subjects:	TP248.13 Biotechnology
Online Access:	http://irep.iium.edu.my/38507/ http://irep.iium.edu.my/38507/ http://irep.iium.edu.my/38507/1/May_Dayah_JPAM.pdf

id	iium-38507
recordtype	eprints
spelling	iium-385072018-06-19T07:17:03Z http://irep.iium.edu.my/38507/ Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus Ahmad Hassan, Nurhidayah Amid, Azura Mohd. Salleh, Hamzah TP248.13 Biotechnology VDAC2 protein identified to be over expressed in over-stunned chicken is a potential biomarker in differentiating stunned chicken and the voltage applied during the process. The VDAC2 gene was amplified by proofread DNA polymerase, then purified and inserted into pENTR-TEV-D-TOPO entry vector and transformed into One Shot TOP 10 Chemically Competent E. coli. The entry clone then was sub cloned into pDEST17 expression vector using LR recombinasein E. coli DH5á as the host. The vector construct was transformed into E. coli BL21-AI for protein expression. Recombinant VDAC2 protein was purified by affinity chromatography using Ni-NTA spin-column. Western blot using polyclonal anti-N terminal human VDAC2 and anti-His tag, followed by secondary antibody AP goat anti-rabbit revealed a 34 kDa protein, confirming the expression of recombinant VDAC2. Post-induction temperature, concentration of L-arabinose and postinduction time were selected for optimization studies under Response Surface Methodology (RSM). The optimum predicted cultivation conditions for the maximum expression of recombinant VDAC2 was found to be at 29oC post-induction temperature, 6.8 hour post-induction time and 0.5% (w/v) L-arabinose with a predicted expression of recombinant VDAC2 intensity of 0.459. The experimental expression of recombinant VDAC2 intensity obtained was 0.496, which was very close to the predicted value. The expression of recombinant VDAC2 improved by almost 6-fold after the optimization process. Therefore, RSM is a suitable method for optimizing recombinant VDAC2 expression in E. coli to provide a better understanding of its characteristics and propertiesto become abiomarker in detecting over-stunned chicken. Journal of Pure and Applied Microbiology 2014-05 Article PeerReviewed application/pdf en http://irep.iium.edu.my/38507/1/May_Dayah_JPAM.pdf Ahmad Hassan, Nurhidayah and Amid, Azura and Mohd. Salleh, Hamzah (2014) Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus. Journal of Pure and Applied Microbiology, 8 (s.ed 1). pp. 751-759. ISSN 0973-7510 http://www.microbiologyjournal.org/
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	TP248.13 Biotechnology
spellingShingle	TP248.13 Biotechnology Ahmad Hassan, Nurhidayah Amid, Azura Mohd. Salleh, Hamzah Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
description	VDAC2 protein identified to be over expressed in over-stunned chicken is a potential biomarker in differentiating stunned chicken and the voltage applied during the process. The VDAC2 gene was amplified by proofread DNA polymerase, then purified and inserted into pENTR-TEV-D-TOPO entry vector and transformed into One Shot TOP 10 Chemically Competent E. coli. The entry clone then was sub cloned into pDEST17 expression vector using LR recombinasein E. coli DH5á as the host. The vector construct was transformed into E. coli BL21-AI for protein expression. Recombinant VDAC2 protein was purified by affinity chromatography using Ni-NTA spin-column. Western blot using polyclonal anti-N terminal human VDAC2 and anti-His tag, followed by secondary antibody AP goat anti-rabbit revealed a 34 kDa protein, confirming the expression of recombinant VDAC2. Post-induction temperature, concentration of L-arabinose and postinduction time were selected for optimization studies under Response Surface Methodology (RSM). The optimum predicted cultivation conditions for the maximum expression of recombinant VDAC2 was found to be at 29oC post-induction temperature, 6.8 hour post-induction time and 0.5% (w/v) L-arabinose with a predicted expression of recombinant VDAC2 intensity of 0.459. The experimental expression of recombinant VDAC2 intensity obtained was 0.496, which was very close to the predicted value. The expression of recombinant VDAC2 improved by almost 6-fold after the optimization process. Therefore, RSM is a suitable method for optimizing recombinant VDAC2 expression in E. coli to provide a better understanding of its characteristics and propertiesto become abiomarker in detecting over-stunned chicken.
format	Article
author	Ahmad Hassan, Nurhidayah Amid, Azura Mohd. Salleh, Hamzah
author_facet	Ahmad Hassan, Nurhidayah Amid, Azura Mohd. Salleh, Hamzah
author_sort	Ahmad Hassan, Nurhidayah
title	Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
title_short	Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
title_full	Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
title_fullStr	Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
title_full_unstemmed	Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus
title_sort	optimization of cultivation conditions to enhance the expression of recombinant vdac2 from gallus gallus
publisher	Journal of Pure and Applied Microbiology
publishDate	2014
url	http://irep.iium.edu.my/38507/ http://irep.iium.edu.my/38507/ http://irep.iium.edu.my/38507/1/May_Dayah_JPAM.pdf
first_indexed	2023-09-18T20:55:20Z
last_indexed	2023-09-18T20:55:20Z
_version_	1777410266467663872

Optimization of cultivation conditions to enhance the expression of recombinant VDAC2 from gallus gallus

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