Construction, purification and characterization of untagged human liver alanine-glyoxylate aminotransferase expressed in Escherichia coli.
His-tagging is commonly used to aid and expedite the purification of recombinant proteins. It is commonly assumed, though less frequently tested, that the His-tag affects neither the structure nor the stability of the protein. Alanine:glyoxylate aminotransferase (AGT) is a peroxisomal pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the transamination of alanine and glyoxylate to pyruvate and glycine. AGT is a clinically relevant enzyme whose deficiency causes an inherited rare metabolic disorder named primary hyperoxaluria type I. Until now, the structure and function of this enzyme have been studied using recombinant wild-type AGT and variants purified using a hexa-histidine tag. However, the study of the functional roles of the N- and C-termini in the dimerization process and on the import into the peroxisome, respectively, requires the preparation of human liver AGT without histidine tags. We report for the first time the expression of untagged AGT together with a new rapid protocol for its purification. In addition, the kinetic parameters for the forward and reverse transamination catalyzed by untagged AGT as well as the spectroscopic features, the K(D(PLP)), the pH and thermal stability of the enzyme in the holo- and apo-form have been determined. This investigation will be the starting point for a detailed understanding of the contributions of the N- and C-termini on the dimerization and folding of AGT, and on its import into the peroxisome. This is prerequisite to understand how pathological mutations affect the proper native quaternary and tertiary structure, stability, and targeting of the enzyme.
Cellini
Barbara
B
Dipartimento di Scienze Morfologico-Biomediche, Università degli Studi di Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Montioli
Riccardo
R
Bianconi
Silvia
S
López-Alonso
Jorge P
JP
Voltattorni
Carla Borri
CB
D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase