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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Journal of Pure and Applied Microbiology
2014
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| Subjects: | |
| 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 |
| Summary: | 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 DH5รก 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. |
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