Overexpression and purification of asparagine synthetase from Escherichia coli.
Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.
Institute for Chemical Research, Kyoto University.
A
A
Sugiyama
H
H
Kato
T
T
Nishioka
J
J
Oda
D000595:Amino Acid Sequence; D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D001217:Aspartate-Ammonia Ligase