RESUMO
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.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas/biossíntese , Escherichia coli/genética , Exotoxinas/biossíntese , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência , Sequência de Aminoácidos , Aminoácidos/análise , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade , Meios de Cultura , Dissulfetos/química , Escherichia coli/crescimento & desenvolvimento , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/isolamento & purificação , Expressão Gênica , Regulação da Expressão Gênica/genética , Focalização Isoelétrica , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/química , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tripsina/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
The solid-phase synthesis of the N alpha-Fmoc analog of protein kinase C substrate (PKCS, Lys-Arg-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg) was characterized by low recovery from the resin and the concomitant appearance of four impurities. FAB-MS revealed molecular weights for two of these impurities that corresponded to the desired peptide plus Tos or Bzl. The other two were justified by invoking a CO2 elimination of the Clz protecting group to yield: 1) peptide plus 2-chlorobenzyl (ClBzl) and 2) peptide plus ClBzl and Tos. A CF-FAB analysis of carboxypeptidase digestions allowed observation of peptide cleavage down to an ion corresponding to lysine, Fmoc, and the corresponding protecting group(s). These data revealed that the impurities were not the result of incomplete deprotection but the result of migration of the protecting groups to the N-terminal end of the peptide. NMR experiments were subsequently performed and revealed the exact site of substitution: the meta positions of the N-terminal Fmoc. These impurities are presumed to arise by electrophilic aromatic substitution of the fluorene group during HF treatment. The desired Fmoc analog served as a convenient, albeit low-yielding, intermediate for purification of the highly charged PKCS by preparative self-displacement HPLC.
