Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase.
An improved method for the production, cleavage, and purification of fusion proteins and peptides is described. The unique aspect of this method is dependent on the use of a proteinase from tobacco etch virus (TEV). The proteinase used is a recombinant TEV proteinase produced with a polyhistidine tract positioned at the amino terminus. The proteinase recognizes a specific, extended cleavage site sequence. The peptide or protein of interest is purified as a fusion protein with a TEV proteinase cleavage site sequence located between it and an affinity carrier portion of the fusion. Incubation with the recombinant TEV proteinase mediates release of the peptide or protein of interest. Use of the recombinant TEV proteinase to cleave fusion proteins is an improvement over use of other proteinases for several reasons, including its high degree of specificity, its insensitivity to many proteinase inhibitors generally used in protein purification, and the ready separation of both the affinity tag and the proteinase from the cleaved product of interest.
Parks
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TD
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Leuther
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Howard
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Johnston
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SA
Dougherty
W G
WG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel; D010455:Peptides / Q000378:metabolism*; D010942:Plant Viruses / Q000201:enzymology*; D010957:Plasmids; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012685:Sepharose; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D009532:Nickel; D012685:Sepharose; D005982:Glutathione Transferase; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses; D005978:Glutathione