Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression
Journal Title: American Journal of Biochemistry and Biotechnology - Year 2006, Vol 2, Issue 4
Abstract
Soybean mosaic virus (SMV)-CN18 is an Rsv resistance-breaking (RB) isolate to overcome soybean resistance genes Rsv1, Rsv3 and Rsv4. The aim of this study was to characterize nuclear inclusion protein a (NIa protein) of RB isolate at the molecular level and demonstrate its processing into genome-linked protein (VPg) and NIa-Pro domains in Esherichia coli containing a bacterial expression pET vector inserted with NIa gene. The full-length of NIa gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) and its 1298 nucleotides (nt) and 432 amino acids (aa) were deduced. The nt and aa sequences of NIa gene of SMV-CN18 shared high identities with the corresponding sequences of the NIa gene of the known SMV isolates, suggesting that the NIa is a highly conserved protein. The NIa-Pro domain contains a highly conserved structural motif for proteolysis, while the VPg domain contains a nuclear localization signal (NLS), a putative NTP-binding site and cellular factor-binding sites. The phylogenetic tree revealed that less divergence of NIa protein exists among twelve SMV isolates, which can be supported by a low bootstrap value between clades. In addition, the full-length of NIa gene, amplified by RT-PCR, was ligated into pET-28b E. coli expression vector with an N-terminal His6-tag. Optimal conditions for expression were at 1mM treatment of IPTG at 25°C for 5 hr. The released protein from bacterial lysates remained soluble and proved the processing form of the NIa polyprotein. E. coli expression system shows the processed product of 29 kDa VPg in SDS-PAGE confirmed by western blot analysis in both crude extracts and purified elution products, using Ni 2+ -NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.
Authors and Affiliations
Bong Kum Choi
Static and Dynamic Mechanical Behavior of Hydroxyapatite-Polyacrylic Acid Composites Under Simulated Body Fluid
In this work, we have investigated mechanical response of hydroxyapatite/polyacrylic composites under dry, wet and simulated body fluid conditions. Hydroxyapatite (HAP) is mineralized under two conditions; one, in pres...
Fabrication of 3-D Porous Mg/Zn doped Tricalcium Phosphate Bone-Scaffolds via the Fused Deposition Modelling
Three dimensionally interconnected porous resorbable beta-tricalcium phosphate (-TCP) ceramic scaffolds were developed using the indirect fused deposition modelling process. -TCP was doped with Mg and Zn, separately,...
Effective Thermophilic Composting of Crop Residues for Inactivation of Tobacco Mosaic Virus
An effective thermophilic composting bioreactor, in which a homogenous distribution of temperature was maintained at 63-65°C by the addition of a bioavailable carbon and low mixing, was developed. The bioreactor operat...
Nanocrystalline Powders of Alkaline-Earth Phosphates as Precursors for Bioceramics
Bone is a nanocomposite made of calcium phosphates and collagen. Collagen has a typical fibrous structure, with diameter ranging from 100 nm to 2 µm. It is suggested that calcium hydroxyapatite, Ca10(PO4)6(OH)2 in the...
Control of Phenolic Compound Secretion and Effect of Growth Regulators for Organ Formation from Musa spp. cv. Kanthali Floral Bud Explants
The study was undertaken to examine banana cv. Kanthali floral bud apex as an alternative source material for in vitro propagation because huge number of explants die due to microbial contamination in case of shoot tip...