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Page No. : 21-25

ABSTRACT

Higher pandemic potential of emerging and re-emerging zoonotic agents poses a significant challenge to public health therefore their timely diagnosis is very important. With the advancements in recombinant DNA technology, the utilization of specific recombinant proteins in development of diagnostic tests offers a high level of diagnostic sensitivity and specificity. Additionally, the method used for protein purification substantially affects the purity, yield and concentration of recombinant protein obtained. Therefore, the present study was undertaken to compare two methods of recombinant protein elution namely imidazole gradient and pH gradient using Immobilized Metal Affinity Chromatography (IMAC) for protein purification. After successful expression and bulk production of recombinant NS1 protein of Japanese Encephalitis virus (JEV), it was purified by IMAC using Imidazole gradient method and pH gradient method for protein elution under denaturing conditions followed by dialysis in decreasing concentration of urea in PBS. The concentration of protein was measured using Bradford reagent. The rNS1 protein eluted by imidazole gradient method was having concentration of 700 μg/ml whereas with pH gradient method it was 1mg/ml. Storage life of recombinant protein was assessed by re-checking the protein concentration after 15 and 30 days of storage at -80OC. Concentration of rNS1 protein eluted with imidazole gradient was 700 μg/ml after 15 days of storage, as recorded previously while it was reduced to 600 μg/ml from 1 mg/ml with pH gradient method. Similarly, storage for one month further reduced the concentration of protein purified by pH gradient method to 350 μg/ml, which reveal the low stability and less storage life of recombinant protein purified by pH gradient method. Further studies are needed to know the exact reason behind the low stability of protein purified using pH gradient method. Albeit our study concludes that stability of recombinant protein after purification must be analysed for development of robust diagnostics.

Keywords: Antigen stability, protein purification, recombinant protein