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...

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Bibliographic Details
Published inJournal of protein chemistry Vol. 11; no. 5; p. 467
Main Authors Kan, C C, Gehring, M R, Nodes, B R, Janson, C A, Almassy, R J, Hostomska, Z
Format Journal Article
LanguageEnglish
Published United States 01.10.1992
Subjects
Acyltransferases - genetics
Acyltransferases - isolation & purification
Acyltransferases - metabolism
Amino Acid Sequence
Asparagine - chemistry
Bacteriophage T7 - genetics
Base Sequence
Carbon-Nitrogen Ligases
Escherichia coli - enzymology
Escherichia coli - genetics
Gene Expression
Histidine - chemistry
Humans
Hydroxymethyl and Formyl Transferases
Molecular Sequence Data
Multienzyme Complexes
Mutagenesis, Site-Directed
Phosphoribosylglycinamide Formyltransferase
Promoter Regions, Genetic
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
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Summary: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.
ISSN:0277-8033
DOI:10.1007/BF01025023