Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein.
Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmaceutical Research and Development, Genetech, Inc., South San Francisco, California 94080.
S J
SJ
Shire
P
P
McKay
D W
DW
Leung
G J
GJ
Cachianes
E
E
Jackson
W I
WI
Wood
K
K
Raghavendra
L
L
Khairallah
T M
TM
Schuster
D000107:Acetylation; D001483:Base Sequence; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011487:Protein Conformation; D012367:RNA, Viral / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000235:genetics / Q000648:ultrastructure; D014026:Nicotiana / Q000382:microbiology; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000648:ultrastructure*
D036022:Capsid Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; C009496:H protein, Tobacco mosaic virus; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins