<abstract xml:lang="en" xmlns="http://www.w3.org/1999/xhtml"><p>The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several <italic>E. coli</italic> host cells. For protease and RdRP, the best expression system containing pColdI vector and <italic>E. coli</italic> WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and <italic>E. coli</italic> BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.</p></abstract>

The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several E. coli host cells. For protease and RdRP, the best expression system containing pColdI vector and E. coli WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and E. coli BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.

Expression and characterisation of the ryegrass mottle virus non-structural proteins

Latvian Biomedical Research and Study Centre, Ratsupītes iela 1, Rīga, LV-1067, LATVIA

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes...

Expression and characterisation of the ryegrass mottle virus non-structural proteins

Pubblicato online: 09 ago 2011

Pagine: 215 - 222

DOI: https://doi.org/10.2478/v10046-010-0035-4

Parole chiave
plant, Ryegrass mottle virus, VPg, P16 protein

This content is open access.

Expression and characterisation of the ryegrass mottle virus non-structural proteins

Pubblicato online: 09 ago 2011

Pagine: 215 - 222

DOI: https://doi.org/10.2478/v10046-010-0035-4

Parole chiaveplant, Ryegrass mottle virus, VPg, P16 protein

This content is open access.

Parole chiave
plant, Ryegrass mottle virus, VPg, P16 protein