Methyltransferase Activity of Dengue RNA Virus by Using Purified Protein

Dengue disease (DENV) is the purpose behind dengue fever. It is a mosquito-borne single positive-stranded RNA contamination of the family Flaviviridae; sort Flavivirus. Dengue illness is a mosquito-borne making or re-rising pathogen. Its positive sense RNA genome has a top at the 5'end and no poly(A) tail at the 3'end. The viral RNA encodes a single polyprotein, C-M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is prepared into three auxiliary proteins (C, M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional chemicals performing different undertakings in the viral life cycle. The N-terminal area of NS5 has unmistakable GTP and S-adenosylmethionine (SAM) restricting locales. The job of GTP restricting site is trapped in the guanylyltransferase (GTase) movement of NS5. We depict the development of the MTase space of NS5 in an E. coli articulation vector and conditions for enzymatic activity of N7-and 2'O-methyltransferase exercises that yield the last sort I 5'topped RNA. Citation: Alwabli AS, Alattas SG, Alhebshi AM, Zabermawi NM, Alkenani N, et al. (2019) Methyltransferase Activity of Dengue RNA Virus by Using Purified Protein. Int J Drug Dev & Res 11: 16-20 Volume 11(2):16-20 (2019)-017 Int J Drug Dev & Res ISSN: 0975-9344 Cloning of NS5 methyltransferase domain To clone the NS5 methyltransferase gene sequence was retrieved from PubMed and primers were designed to amplify the NS5 methyltransferase region expected PCR size is 786 base pair amplifying NS5 methyltransferase domain for dengue virus independently. NS5 methyltransferase gene contains introns; therefore it was amplified from the cDNA, and cloned cDNA was set up from the culture utilizing fitting unit, and it encodes amino acid 262 amino acid. The purified PCR products were subjected to ligation into the pGEMT easy vector for T-A cloning. The ligated mixture was transformed into DH5α E. coli cells and was placed on LB-Agar containing IPTG and X-gal. The white colonies were screened for positive clone by restriction digestion and by colony PCR as well. After restriction digestion, NS5 methyltransferase gene inserts of positive clones were purified from agarose gel and were subjected to standard ligation into an expression vector (pET28a+) (Novagen, Madison, WI, USA) and then transformed into E. coli DH5α. The plasmids of recombinant colonies were purified and confirmed by restriction digestion analysis. Expression and purification of recombinant protein The pET28a+ expression clones were transformed into E. coli strains BL21 codon plus. 1.0 mM IPTG induced the overexpression of recombinant proteins. The harvested culture pellet was resuspended into bacterial lysis buffer of pH 7.8 (20 mm Tris-HCl, 250 mm NaCl, 0.1% Tween 20, 0.1% Triton 100 and the protease inhibitor cocktail from Sigma (St. Louis, MO, USA) and sonicated to lyse the maximum number of cells. The soluble fraction was separated after centrifugation and allowed to bind with equilibrated Ni-NTA (Qiagen, GmbH, Germany) in the binding buffer (20 mm Tris-HCl pH 8.0, 250 mm NaCl, 5 mM imidazole and protease inhibitor cocktail (Sigma, St. Louis, MO, USA) for one and half hour at 4°C. The column was stringently washed with excess wash buffer (10-20 mm Tris-HCl pH 8.0, 250 mm NaCl, 25-50 mM imidazole and protease inhibitor cocktail) to remove the non-specifically bounded protein from Ni-NTA. The recombinant His-tag protein was eluted with varying concentration of imidazole (50-250 mm) in elution buffer (20 mm Tris-HCl pH 8.0, 250 mm NaCl, 10% (v/v) glycerol and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Purified protein fraction was further subjected to SDS-PAGE and western blot to check the purity of proteins. The purified recombinant proteins were used for the rest of the experiments. Western blot analysis The purified proteins were transferred from the SDS-PAGE gel onto nitrocellulose membrane or PVDF. Transfer of the purified proteins to the membrane was checked by using PonceauS staining before the blocking. The blot was blocked in blocking buffer (3% BSA) for 2, and subsequently, it was washed and incubated for 1 hour with primary antihis tag antibody (1:3000). The blot was then removed and incubated for 1 hour with appropriate secondary antibody (1:5000) conjugated to alkaline phosphatize or horseradish peroxidize (HRP) and incubate at room temperature for 2 hours. The blots were developed by using of ECL kit. The reaction was stopped by rinsing the stain in 10 mm EDTA, pH 8.0.