High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles.
The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs.
Saini
Manisha
M
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, New Delhi 110 067, India.
Vrati
Sudhanshu
S
D001483:Base Sequence; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins