Recombinant RNA phage Q beta capsid particles synthesized and self-assembled in Escherichia coli

Gene. 1993 Dec 27;137(1):133-7. doi: 10.1016/0378-1119(93)90261-z.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allolevivirus / genetics*
  • Amino Acid Sequence
  • Base Sequence
  • Capsid / genetics*
  • Genes, Viral
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • RNA, Viral / genetics*
  • RNA-Directed DNA Polymerase
  • Recombinant Fusion Proteins / genetics*

Substances

  • Qbeta ribonucleic acid
  • RNA, Viral
  • Recombinant Fusion Proteins
  • RNA-Directed DNA Polymerase

Associated data

  • GENBANK/L11589
  • GENBANK/L11590
  • GENBANK/L11591
  • GENBANK/L11592
  • GENBANK/L11593
  • GENBANK/L11594
  • GENBANK/L24529
  • GENBANK/M99039
  • GENBANK/Z15047
  • GENBANK/Z15048