Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli.
The coast protein of alfalfa mosaic virus (AMV) was cloned and expressed in Escherichia coli as a fusion protein containing a 37 amino acid extension with a (His)6 region for affinity purification. About half of the expressed recombinant coat protein (rCP) was soluble upon extraction and half was insoluble in inclusion bodies. Western blot analysis confirmed the identity of the rCP and protoplast infectivity assays indicated that the rCP was biologically active in an early event of AMV infection, called genome activation. The rCP assembled into T = 1 empty icosahedral particles, as described previously for native coat protein. Empty particles formed hexagonal crystals that diffracted X-rays to 5.5 A resolution. The crystals of trypsin-treated particles of rCP appear to be isomorphous with crystals of trypsin-treated particles of native coat protein, Spherical particles containing RNA assembled when the rCP was combined with in vitro transcripts of AMV RNA4, the smallest naturally encapsidated AMV RNA. Bacilliform particles that resembled native virions assembled when the rCP was combined with transcripts of RNA1, the largest genomic RNA.
Yusibov
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Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.
Kumar
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North
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Johnson
J E
JE
Loesch-Fries
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LS
D017794:Alfalfa mosaic virus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D036022:Capsid Proteins; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
D036022:Capsid Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C095691:coat protein, Alfalfa mosaic virus