Ricin toxin is a naturally occurring toxin that is derived from the seeds of Ricinus communis, commonly known as castor beans. It is composed of an enzymatically active cytotoxic polypeptide chain, commonly called the "A" chain and sometimes referred to herein as "RTA", that is bound by a single disulfide link to a second polypeptide chain commonly called the "B" chain that is presumed to be responsible for binding the toxin molecule to cells and aiding in translocating RTA into the cytoplasm. RTA is capable of catalytically inactivating the large subunit of ribosomes in vitro and the mechanism of RTA for in vivo cytotoxicity is believed to reside in this capacity for ribosome inactivation.
Olsnes, S. Perspectives in Toxicology, A. W. Bernheimer, Ed (1977) J. Wiley & Sons, NY, pp 122-147 and Olsnes, S., et al., Molecular Action of Toxins and Viruses, Cohen, et al, Ed (1982) Elsevier, Amsterdam, pp 51-105 characterize native RTA as having an apparent molecular weight of 32,000. Copending commonly owned U.S. patent application Ser. No. 715,934 filed Mar. 25, 1985 discloses the native structural gene for RTA, the deduced amino acid sequence of RTA, DNA constructs for cloning and expressing the RTA gene, and transformed bacteria capable of synthesizing intracellularly produced, soluble recombinant RTA. The patent application further describes the production of such recombinant RTA by such bacteria and a procedure for recovering RTA from the bacteria. The recovery procedure comprises sonicating the cells in an aqueous suspension under reducing conditions at a pH of 8.5, centrifuging the sonicate, and chromatographing the supernatant using a phenylsepharose column to produce a partially purified soluble form of RTA. This RTA was further purified by successive chromatographing on a carboxymethyl cellulose column and a Cibacron Blue F3GA column. While this recovery process provides substantially pure RTA, it is tedious and suffers from low yields.
The present invention provides a simpler procedure for recovering substantially pure, soluble recombinant RTA in higher yields from the transformants.
USPTO Backgrounds
USPTO Backgrounds