THE EXPRESSION, AFFINITY PURIFICATION AND CHARACTERIZATION OF RECOMBINANT PSEUDOMONAS-EXOTOXIN-40 (PE40) SECRETED FROM ESCHERICHIA-COLI

Procedures have been devised for producing high yields of purified rec ombinant PE40, a protein important for the development of the anti-AID S therapeutic, sCD4-PE40. PE40 is a truncated form of the bacterial to xin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but ret ains domains II and III that are involved in membrane translocation an d inhibition of protein synthesis in eukaryotic cells. Expression vect ors in Escherichia coli encoding PE40 synthesized the product as a sol uble protein under control of the T7 promoter. The expression capabili ties of transformants of E. coli BL21(DE3) were highly unstable. Expre ssion levels (secreted and total) were evaluated in shake flasks and a t the 10-1 scale at 27 degrees C and 37 degrees C, and following induc tion by IPTG or lactose. The cell-free media from the batch process wa s applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity. This purific ation strategy was based on the structural similarity of the blue dye to NAD, a natural substrate for domain III of PE40. Green and red dye- ligand chromatography steps removed nicotinamide as well as minor resi dual E. coli proteins from PE40. Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ioniz ation mass spectrometry to determine the sequence and verify disulfide bonding.