Enzyme purification by genetically attached polycysteine and polyphenylalanine affinity tails.
Two novel affinity tails, polycysteine and polyphenylalanine, have been genetically attached to galactokinase (EC 2.7.1.6) and beta-galactosidase (EC 3.2.1.23) in order to facilitate their purification. A chemically synthesized DNA linker encoding four cysteine residues was thus fused in frame with the galactokinase gene. The gene product, cysteine galactokinase, was significantly retarded on a column of thiopropyl-Sepharose. Using pulse elution, cysteine galactokinase was eluted at 10 mM DTT. Under the condition used, native galactokinase did not bind to thiopropyl-Sepharose. Homopolymer tailing was employed to prepare a phenylalanine-modified beta-galactosidase. One of the obtained genetic transformants coding for a beta-galactosidase carrying 11 phenylalanine residues at the N-terminus of the enzyme was isolated. With the aid of hydrophobic interaction chromatography the modified enzyme could be purified to homogeneity on fast protein liquid chromatography using a phenyl-Superose column.
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005686:Galactokinase / Q000302:isolation & purification*; D005696:Galactosidases / Q000302:isolation & purification*; D010455:Peptides; D010957:Plasmids; D012685:Sepharose / Q000031:analogs & derivatives; D001616:beta-Galactosidase / Q000302:isolation & purification*
D010455:Peptides; C012001:polyphenylalanine; C057713:polycysteine; C035872:thiopropyl-sepharose; C033452:Phenyl-Sepharose CL-4B; D012685:Sepharose; D005686:Galactokinase; D005696:Galactosidases; D001616:beta-Galactosidase