Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.
Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.
Bernasconi
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Sandoz Agro Inc., Research Division, Palo Alto, California 94304-1104.
Walters
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Woodworth
A R
AR
Siehl
D L
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Stone
T E
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Subramanian
M V
MV
D000495:Allosteric Site / Q000235:genetics; D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D000878:Anthranilate Synthase