Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina.
Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.
Noda Development Group, Planning & Administration Department, Kikkoman Biochemifa Company, 376-2, Kamihanawa, Nodashi, Chiba, 278-0033, Japan. ernasu@mail.kikkoman.co.jp
Eriko
E
Nasu
Atsushi
A
Ichiyanagi
Keiko
K
Gomi
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000378:metabolism; D015870:Gene Expression; D044129:Halomonadaceae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010756:Phosphoric Acids / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D004952:Esters; D010756:Phosphoric Acids; D011994:Recombinant Proteins; C030242:phosphoric acid; D000469:Alkaline Phosphatase