Expression of the turnip yellow mosaic virus proteinase in Escherichia coli and determination of the cleavage site within the 206 kDa protein.
The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C-terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.
Kadaré
G
G
Institut Jacques Monod, Paris, France.
Rozanov
M
M
Haenni
A L
AL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D001665:Binding Sites; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017806:Tymovirus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases