Expression and purification of a neuropeptide nocistatin using two related plant viral vectors.
Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.
Lim
Aileen A L
AA
Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore 117543, Singapore.
Tachibana
Shinro
S
Watanabe
Yuichiro
Y
Wong
Sek Man
SM
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D018847:Opioid Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018515:Plant Leaves / Q000821:virology; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D011995:Recombination, Genetic; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D017898:Tobamovirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
D018847:Opioid Peptides; C111148:nocistatin