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J Gen Virol 77 (1996), 567-573; DOI 10.1099/0022-1317-77-4-567
© 1996 Society for General Microbiology
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Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli

Vidadi Yusibov1, Abhinav Kumar2,{dagger}, Adam North2, John E. Johnson2,{dagger} and L. Sue Loesch-Fries1,*

1 Department of Botany and Plant Pathology
and2 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

The coat 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 Å resolution. The crystals of trypsin-treated particles of rCP appear to be isomorphous with crystals of trypsintreated 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.

{dagger} Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

* Author for correspondence. Fax +1 317 494 5896. e-mail loeschfries@btny.purdue.edu

Received 14 September 1995; accepted 21 November 1995.


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