Expression, purification, and characterization of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase.
A genetic construct consisting of the synthetic gene coding for human muscle acylphosphatase linked to the gene for glutathione S-transferase has been prepared. This gene was transformed into and expressed by the Escherichia coli strains DB1035 and TB1, respectively. The fusion protein was purified by affinity chromatography and subsequently cleaved to the fully active acylphosphatase, which was further purified by gel filtration chromatography. Such a purification procedure is very rapid and suitable for obtaining considerable amounts of enzyme at a very high yield. The purified human muscle acylphosphatase was fully active and showed structural features, as well as kinetic and stability parameters, identical to those of the native enzyme.
Modesti
A
A
Department of Biochemical Sciences, University of Florence, Italy.
Taddei
N
N
Bucciantini
M
M
Stefani
M
M
Colombini
B
B
Raugei
G
G
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D017931:DNA Primers; D005982:Glutathione Transferase; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000097024:Acylphosphatase