Imperial Chemical Industries Limited

Zeneca Limited

John E Fitton

David Timms

Robert Craig Hockney

Bhuphendra Vallabh Kara

Processes for preparing polypeptides such as ricin A such that soluble polypeptide may be obtained. The pH may be adjusted during the process or the temperature decreased during the terminal portion of the cultivation, and yeast extract may be added to the growth medium during the cultivation. In the case of ricin A the pH is generally adjusted by lowering the pH from a first value such as 6.7 to a second value such as a value between about 5.5 and 6.7.

Fermentation processes for preparing soluble ricin A

Ribotoxins, such as restrictocin, mitogillin and alpha-sarcin which do not contain a cysteine for linkage to antibodies for immunotoxin production need to be modified for such linkage without compromising biological activity. Protein analogues of such native ribotoxins are provided in which both the analogue and the native ribotoxin can cleave only a single phosphodiester bond of 28S rRNA in a 60S ribosomal subunit and in which the analogue comprises only one cysteine available from covalent linkage to a partner, such as an antibody, which cysteine is not present in the native ribotoxin. Especially preferred analogues are restrictocin Cys 13, Cys 82, Cys 106 and Cys 110 and restrictocin Cys 150-Gly151. Corresponding DNA sequences, vectors, host cells, immunotoxins and pharmaceutical compositions are also claimed.

Restrictocin-like ribotoxin analogues comprising only one cysteine

Methods for recombinant production in procayotic microorganisms such as E. coli of ribotoxins such as restrictocin, alpha-sarcin and mitogillin are described. Known methods were relatively low yielding and not cost effective for commercial use such as in the pharmaceutical industry where relatively large quantities of toxin with consistent batch to batch quality may be required for immunotoxin production. Use of recombinant methods of production open up the possibility of making ribotoxin analogues. Toxicity of ribotoxins was recognised as a concern in development of a high yielding cost effective production method. Methods for high yielding intracellular accumulation or secretion of ribotoxins are described. Use of protease deficient strains and other methods of minimising breakdown of ribotoxin by protease are preferred. Vectors and host strains for use in the methods are described.

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Buxton, United Kingdom

John E Fitton