Wood, Katherine Anne (1991) A preproabrin : genomic cloning and expression of the constituent polypeptides in heterologous systems. PhD thesis, University of Warwick.

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Abstract

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 the similarity with the ricin toxins and Ricinus communis agglutinin, the preproprotein 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 eukaryotic ribosomes has been demonstrated in vitro.

The mature B-chain of the preproabrin has been expressed in Xenopus laevis oocytes and was found to be unstable and lacking in lectin activity. The non-lectin activity of the protein was confirmed by expression and analysis of the preproabrin in X. laevis oocytes.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany
Library of Congress Subject Headings (LCSH):	Plant lectins, Plant toxins, Molecular cloning, Gene expression, Immunotoxicology, Nucleotide sequence, Escherichia coli -- Genetics
Official Date:	September 1991
Dates:	Date Event September 1991 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Lord, Mike (J. Mike)
Sponsors:	Science and Engineering Research Council (Great Britain)
Format of File:	pdf
Extent:	273 leaves : illustrations, charts
Language:	eng

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