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Authors :-Sneha Rao, Sudarson Sundarrajan, Sriram Padmanabhan

Volume/Issue:  Volume 2 Issue 9

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 https://goo.gl/DF9R4u

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Reverse transcriptase (RT) from Moloney murine leukemia virus (MMuLV) is an RNA-dependent DNA polymerase that uses single-stranded RNA, DNA, or an RNA-DNA hybrid to synthesize a cDNA strand. The purification methods available for this enzyme are laborious and not cost-effective. We present a novel and efficient protein refolding protocol for solubilization and purification of the catalytic fragment of MMuLV-RT. Our protocol is based on the denaturation of the RT enriched inclusion bodies with an alkaline solution of 8 M urea, followed by refolding in a refolding buffer and a single buffer-exchange steps via dialysis. The protocol proved effective for us to obtain a large amount of relatively pure and rightly folded RT protein that exhibited potent reverse transcriptase activity on HIV and HCV viral RNA’s comparable to commercially available MMuLV RT enzyme preparation. The yield of ~23 mg of purified RT/litre of E. coli culture is the highest yield reported to date and the purification process presented in this article is scalable with high recovery rate.
Keywords— MMuLV Reverse Rranscriptase; Protein Refolding; Ni-NTA Chromatography; HCV Diagnosis; HIV Diagnosis.