Research Article Open Access

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Bong K. Choi1, Jae S. Park1, Hye J. Ahn1, Hye J. Yum1, Vasudevan Ayyappan1, Lakshmi S. Vijayachandran1, Ji S. Kim1, Sei C. Kim1 and Chang W. Choi1
  • 1 Pai Chai University, Korea

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 Ni2+-NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.

American Journal of Biochemistry and Biotechnology
Volume 2 No. 4, 2006, 148-153

DOI: https://doi.org/10.3844/ajbbsp.2006.148.153

Submitted On: 18 October 2006 Published On: 31 December 2006

How to Cite: Choi, B. K., Park, J. S., Ahn, H. J., Yum, H. J., Ayyappan, V., Vijayachandran, L. S., Kim, J. S., Kim, S. C. & Choi, C. W. (2006). Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression. American Journal of Biochemistry and Biotechnology, 2(4), 148-153. https://doi.org/10.3844/ajbbsp.2006.148.153

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Keywords

  • Soybean mosaic virus (SMV)
  • Rsv resistance-breaking (RB) isolate
  • nuclear inclusion protein a (NIa)
  • processing
  • genome-linked protein (VPg)
  • NIa-Pro
  • highly conserved protein
  • E. coli expression