Recombinant RNA phage Q beta capsid particles synthesized and self-assembled in Escherichia coli.
The Escherichia coli RNA phage Q beta coat protein-encoding gene (C) was amplified from native Q beta RNA using a reverse transcription-PCR technique. Gene C contains sequences coding for both the 133-amino acid (aa) Q beta coat protein (CP) and the 329-aa read-through protein (A1) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon. Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared. An amplified 1062-bp PCR fragment was positioned under the control of the strong E. coli trp promoter (Ptrp) within a pGEM-derived plasmid. The synthesis of gene C products was confirmed electrophoretically and immunologically. An immunodiffusion test with anti-Q beta phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Q beta C gene was responsible for high-level synthesis and correct self-assembly of Q beta CP monomers into capsids indistinguishable morphologically and immunologically from Q beta phage particles, which we plan to use as surface display vectors.
Kozlovska
T M
TM
Institute of Molecular Biology, Latvian Academy of Sciences, Riga.
Cielens
I
I
Dreilinņa
D
D
Dislers
A
A
Baumanis
V
V
Ose
V
V
Pumpens
P
P
D017908:Allolevivirus / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D011993:Recombinant Fusion Proteins / Q000235:genetics*
C028696:Qbeta ribonucleic acid; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012194:RNA-Directed DNA Polymerase