Immobilization and single-step purification of fusion proteins using DEAE-cellulose.
We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain--beta-galactosidase chimera, which can be purified by this procedure and shows a high beta-galactosidase activity when immobilized in the column. A vector plasmid, pCUZ1, containing the lppp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-beta-D-galactoside procedure. A chimera between the choline-binding domain and the pneumococcal hemolysin was also constructed and purified using pCUZ1.
Sanchez-Puelles
J M
JM
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Sanz
J M
JM
Garcia
J L
JL
Garcia
E
E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western; D002794:Choline / Q000235:genetics / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013301:Streptolysins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D001616:beta-Galactosidase; D002794:Choline