Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase.
A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.
Hellman
J
J
Centre for Biotechnology, Turku, Finland.
Paavilainen
S
S
Mäntsälä
P
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003593:Cytoplasm / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010957:Plasmids; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase