Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase.
The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.
Lee
Peychii
P
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Colman
Roberta F
RF
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000264:Adenylosuccinate Lyase / Q000096:biosynthesis* / Q000302:isolation & purification*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013696:Temperature
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C012168:adenylosuccinate; D000249:Adenosine Monophosphate; D000264:Adenylosuccinate Lyase