A rapid method for the purification of large amounts of an alpha-complementing peptide derived from beta-galactosidase (E. coli).
A DNA segment coding for residues 6-44 of beta-galactosidase was ligated to a vector with the glutathione-S-transferase gene which also contained a sequence coding for a thrombin recognition site. The fused protein, with an additional 9 amino acids coded for by the vector, was purified using a glutathione agarose affinity column. A peptide made up of residues 6-44 of beta-galactosidase and the 9 additional amino acids was then cleaved from the glutathione-S-transferase using thrombin and purified with a gel filtration column. The peptide was about 3-4 times as active for alpha-complementation as the alpha-peptide derived from CNBr digestion of wild type beta-galactosidase.
Gallagher
C N
CN
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Roth
N J
NJ
Huber
R E
RE
D001426:Bacterial Proteins / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D001616:beta-Galactosidase