May, J. Michael J (1988) The expression of biologically active recombinant ricin A chain "in vitro". PhD thesis, University of Warwick.

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Abstract

The major aim of this project was to attempt to define residues In rlcln A chain which are involved In the catalytic activity of the protein and to define a rlcin A chain molecule of minimum size which still remains active.
A simple and sensitive system was developed in which the expression and assessment of biological activity of recombinant rlcln A chain are combined. This represents one of few reported examples of the ability to assess the activity of protein expressed from in vitro synthesised RHA in a cell free system. When recombinant ricln A chain transcripts were translated in a rabbit reticulocyte lysate, the ribosomes were rapidly inactivated. In contrast, ribosomes which have translated transcripts encoding non toxic polypeptides such as ricin B chain are not inactivated. Ribosome inactivation was accompanied by a highly specific modification of 28S rRNA which Is thought to cause the inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under conditions which inhibit reticulocyte ribosomes, confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inactivation by ricln A chain than are mammalian ribosomes.
Using the same system, it was shown that by deleting an internal hexapeptide which shares homology with hamster EF 2, catalytic activity was completely abolished. Deleting a second Internal pentapeptide, conserved between ricin A chain and trichosanthln, had no effect. Deleting the first nine residues from the V terminus of ricln A chain did not affect toxicity, whereas deleting a further three residues Inactivated the polypeptide. Point mutations which Individually converted arginine 48 and arginine 56 to alanine residues or which removed arginine 56 were also without effect on the catalytic activity of the toxin.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany
Library of Congress Subject Headings (LCSH):	Ricin, Plant toxins, Gene expression, Proteins -- Synthesis
Official Date:	November 1988
Dates:	Date Event November 1988 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Publication Status:	Unpublished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).Biotechnology Directorate
Extent:	1 volume (variouis pagings): illustrations.
Language:	eng

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