Overexpression of human alanine:glyoxylate aminotransferase in Escherichia coli: renaturation from guanidine-HCl and affinity for pyridoxal phosphate co-factor.
Alanine:glyoxylate aminotransferase-1 (AGT) is a human liver peroxisomal enzyme whose deficiency results in, primary hyperoxaluria type 1 (PH1), a fatal metabolic disease. AGT requires a pyridoxal phosphate (PLP) co-factor in its active site. The AGT gene usually exists in one of two polymorphic forms, the major and minor alleles. We describe here an overexpression system for normal and mutant variants of human AGT in Escherichia coli BL21 (DE3) pLysS. We have extracted functional AGT from inclusion bodies using guanidine-HCl. Denaturation and re-folding of the overexpressed AGT after guanidine-HCl treatment produces high yields of biologically active protein and provides a strategy for generating an apoenzyme to investigate PLP-binding. K(M)s for PLP were determined by reconstitution of the apoenzyme. Successful folding was independent of the presence of PLP. The K(M) for PLP for minor allele AGT was significantly higher than that for major allele AGT. This decreased affinity could be attributed to I340M, a polymorphism associated with the minor allele. G170R, located on the minor allele and the most common PH1 mutation, had no effect on the affinity for PLP. PH1 mutations, G41V and G41R, showed enhanced activity after re-folding. We suggest that the renaturation/re-folding and reconstitution strategies provide an approach for studying the maturation of AGT under optimal conditions and in isolation from cellular quality control and chaperoning processes. Furthermore, our data show that mutations with serious consequences in vivo may not be inherently catalytically inactive and may be rescuable.
Coulter-Mackie
Marion B
MB
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V6H 3N1. marioncm@interchange.ubc.ca
Lian
Qun
Q
Wong
Steve G
SG
D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019791:Guanidine; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D020673:Protein Renaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase; D019791:Guanidine