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 by in vitro mutagenesis, which can improve the pharmacological properties and antitumor activity. Earlier methods of expression strategies using Escherichia 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, a NdeI 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
B Y
BY
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Frankel
A E
AE
Ramakrishnan
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012276:Ricin / Q000235:genetics* / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D012276:Ricin