Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli.
Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A-chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences. Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A-chain of 251 amino acids preceded by 34 amino acids containing an N-terminal signal peptide, followed by a 14-amino-acid linker and a B-chain of 263 amino acids. The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro.
Wood
K A
KA
Department of Biological Sciences, University of Warwick, Coventry, England.
Lord
J M
JM
Wawrzynczak
E J
EJ
Piatak
M
M
D000036:Abrin / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015698:Genomic Library; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D037121:Plant Lectins; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D037121:Plant Lectins; D011498:Protein Precursors; D011994:Recombinant Proteins; C069393:preproabrin; D000036:Abrin