Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X55667. The novel amino acid sequence data have been deposited with the EMBL sequence data bank.
Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli
Article first published online: 3 MAR 2005
European Journal of Biochemistry
Volume 198, Issue 3, pages 723–732, June 1991
How to Cite
WOOD, K. A., LORD, J. M., WAWRZYNCZAK, E. J. and PIATAK, M. (1991), Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli. European Journal of Biochemistry, 198: 723–732. doi: 10.1111/j.1432-1033.1991.tb16072.x
- Issue published online: 3 MAR 2005
- Article first published online: 3 MAR 2005
- (Received January 25/February 27, 1991) – EJB 91 0142
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.