Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence.
The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.
Westby
M
M
Department of Biological Sciences, University of Warwick, Coventry, U.K.
Argent
R H
RH
Pitcher
C
C
Lord
J M
JM
Roberts
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000235:genetics; D008040:Genetic Linkage / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects; D012276:Ricin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000235:genetics; D014981:Xenopus
D004220:Disulfides; D046911:Macromolecular Substances; D011498:Protein Precursors; D011994:Recombinant Proteins; C078236:proricin; D004247:DNA; D012276:Ricin; D010450:Endopeptidases