High-level expression and simplified purification of recombinant ricin A chain

Ricin toxin is a glycoprotein which catalytically inactivates eukaryotic ribosomes by depurination of a single adenosine residue from the 28S ribosomal RNA. The enzymatic activity is present in the A chain of the toxin molecule, whereas the B chain contains two binding sites for galactose. Since it is highly potent in inhibiting protein synthesis, the A chain is used to prepare cytotoxic conjugates effective against tumor cells. Such chimeric proteins are highly selective and have a wide range of clinical applications. Extensive preclinical studies on these conjugates require large amounts of purified A chain. Native ricin A chain is heterogeneous, since plants produce a number of isoforms of ricin toxin. Purified, native preparations often contain two types of ricin A chain which differ in the extent of glycosylation. By cloning and expressing the gene of A chain, one could obtain homogeneous toxin molecules devoid of carbohydrates. In addition, structural changes in the toxin polypeptide could be introduced byin vitro mutagenesis, which can improve the pharmacological properties and antitumor activity. Earlier methods of expression strategies usingEscherichia coli have yielded only moderate levels of expression. In the present study, the coding region of ricin A chain was cloned into pET3b, a high-level expression vector under the control of the T7 promoter. Recombinant ricin A chain produced by this construct has an additional 14 amino acid residues at the NH₂ terminus. Subsequently, aNdeI site was created at the 5′ end of the gene by oligonucleotide-directed mutagenesis. The modified fragment was then introduced into pET3b vector to produce toxin polypeptide identical to the native sequence. The recombinant ricin A chain containing additional residues at the NH₂ terminus was, however, equally effective in inhibiting protein synthesis. This molecule also readily reassociated with the native B chain to produce whole ricin. In the present expression system, almost eightfold higher amounts of the recombinant protein were produced when compared to other constructs. Furthermore, modifications at the C-terminal end, such as the addition of pentalysine, did not affect the expression levels. This indicated that the expression system is suitable for producing mutant forms of ricin A chain as well. Finally, the purification scheme was simplified to obtain large amounts of pure toxin polypeptides.