Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails.
In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.
Lilius
G
G
Pure and Applied Biochemistry, Chemical Centre, University of Lund, Sweden.
Persson
M
M
Bülow
L
L
Mosbach
K
K
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004533:Egtazic Acid; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D005691:Galactose Dehydrogenases / Q000235:genetics / Q000302:isolation & purification*; D006639:Histidine; D008969:Molecular Sequence Data; D010455:Peptides; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification*; D015183:Restriction Mapping; D015032:Zinc / Q000494:pharmacology
D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D004533:Egtazic Acid; D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase; D015032:Zinc