Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography.
In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.
Hwang
Seon-Kap
SK
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Salamone
Peter R
PR
Kavakli
Halil
H
Slattery
Casey J
CJ
Okita
Thomas W
TW
D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D051599:Glucose-1-Phosphate Adenylyltransferase; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology*
D003470:Culture Media; D009841:Oligonucleotides; C033223:polyhistidine; D006639:Histidine; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase