Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain.
We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced in E. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.
Kan
C C
CC
Agouron Pharmaceuticals, Inc., San Diego, California 92121.
Gehring
M R
MR
Nodes
B R
BR
Janson
C A
CA
Almassy
R J
RJ
Hostomska
Z
Z
D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D051026:Phosphoribosylglycinamide Formyltransferase; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006639:Histidine; D001216:Asparagine; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D019731:Carbon-Nitrogen Ligases; C494823:GART protein, human