Expression of recombinant voltage dependant anion channel 2 (VDAC2) protein from electrically stimulated chicken

Abstract

VDAC2 protein identified to be highly expressed in stunned chicken thus can be a potential biomarker in a rapid test detection kit for over stunned chicken. Thus, to obtain this protein, it is cloned and expressed in BL21-AI Escherichia coli. cDNA was synthesized from mRNA of stunned chicken's skeletal muscle and cloned using recombination reaction. All positive clones were identified by PCR and restriction enzyme digestion as well as DNA sequencing. Recombinant VDAC2 protein was purified by Ni-NTA spin-column. Western blot performed using polyclonal anti-N terminal human VDAC2 and anti-His tag revealed a 34 kDa protein, confirming the expression of recombinant VDAC2. The optimum culture conditions such as postinduction temperature, concentration of inducer and post-induction time for the highest expression of VDAC2 were determined from an experimental design using response surface methodology (RSM). The optimum predicted cultivation conditions from the developed model were 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 of 0.46 (OD). The OD is based on the highest calibrated pixel intensities in the spot from which the background has been withdrawn. The background is defined as the minimum pixel value in the spot neighborhood. The experimental expression of recombinant VDAC2 obtained was 0.50 (OD), which was very close to the predicted value which is 0.46 (OD). The expression of recombinant VDAC2 has improved about 6-fold after the culture conditions were optimized. Therefore from this study, chicken VDAC2 has been successfully cloned and expressed.