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 recombinant PE40, a protein important for the development of the anti-AIDS therapeutic, sCD4-PE40. PE40 is a truncated form of the bacterial toxin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but retains domains II and III that are involved in membrane translocation and inhibition of protein synthesis in eukaryotic cells. Expression vectors in Escherichia coli encoding PE40 synthesized the product as a soluble protein under control of the T7 promoter. The expression capabilities of transformants of E. coli BL21(DE3) were highly unstable. Expression levels (secreted and total) were evaluated in shake flasks and at the 10-1 scale at 27 degrees C and 37 degrees C, and following induction by IPTG or lactose. The cell-free media from the batch process was applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity. This purification 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 residual E. coli proteins from PE40. Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ionization mass spectrometry to determine the sequence and verify disulfide bonding.
Kawooya
J K
JK
Upjohn Company, Kalamazoo, MI 49001, USA.
Treat
J C
JC
Kirschner
R J
RJ
Sears
M W
MW
Gorczany
J F
JF
Grode
S H
SH
Strother
D S
DS
Asmus
P A
PA
Eckenrode
F M
FM
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003470:Culture Media; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005098:Exotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000235:genetics; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000596:Amino Acids; D001427:Bacterial Toxins; D003470:Culture Media; D004220:Disulfides; D005098:Exotoxins; D010455:Peptides; D011994:Recombinant Proteins; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A