Cloning, expression, purification and activation by Na ion of halophilic alkaline phosphatase from moderate halophile Halomonas sp. 593.
We have succeeded in the cloning of alkaline phosphatase gene, haalp, from moderate halophile Halomonas sp. 593. A deduced amino acid sequence showed a high ratio of acidic to basic amino acids, characteristic of halophilic proteins. The gene product was efficiently expressed in Escherichia coli BL21 Star (DE3) pLysS, but in an inactive form. The purified recombinant HaALP was separated into four fractions by gel filtration. When they were dialyzed against 50 mM Tris-HCl (pH 8.0)/2 mM MgCl₂ buffer containing 3 M NaCl, one of these four fractions was activated to almost full activity. This fraction contained a folding intermediate that was converted to the native structure by the salt. Among the additional salts tested, i.e., KCl, KBr, LiCl, MgCl₂, (NH₄)₂SO₄, and Na₂SO₄, only Na₂SO₄ was effective, suggesting the importance of Na ion.
Ishibashi
Matsujiro
M
Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. matu@ms.kagoshima-u.ac.jp
Oda
Kazuki
K
Arakawa
Tsutomu
T
Tokunaga
Masao
M
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002414:Cations, Monovalent; D003001:Cloning, Molecular; D004789:Enzyme Activation; D020632:Halomonas; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012965:Sodium Chloride
D001426:Bacterial Proteins; D002414:Cations, Monovalent; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000469:Alkaline Phosphatase