DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter P ~25 fused with the lac operator of E.coli. When induced, E.coZi 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococoal protein A (PA) have been produced in E. coii. Construcb consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specitically depurinating 28 S ribosomal RNA (via RTA). Howevtx, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short ammo acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a dis~~de-links loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA Iinked by a d&hide bond to PA. The cleaved fusion protein was highly toxic to Saudi cells coated with ~~-~~~oglob~in antibody suiting that the RTA could be released from the PA by reduction witbin the oytosol.Ricin A chain; Fusion protein; Protein A, ~oteol~ti~ily-cl~~ble sequexe

DNA encoding Ricinus comtunis agglutinin A chain was ligated into the E. co/i expression VCCIO~ pDS 513. Induced E. coii 71.18 cells which had been transformed with this plasmid express Ricitrus cottrtmmis agglutinin A chain in M soluble and biologically active form. Recombinant Riclnur cmtmnis agglutinin A chain had ribosomal RNA N.glycosidase activity and was approximately IO-fold less active than ricin A chain in a cell-free protein synthesis inhibition assay.Rihtrs cotnmcnis agglutinin A chain; Ricin A chain; Expression; E. co/i