Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system.
Human placental alkaline phosphatase was successfully cloned in the yeast system Pichia pastoris. The recombinant enzyme was over-expressed as a secreted protein in the cultured medium. The enzyme was extremely stable, which resulted in a total recovery of the enzyme activity after the purification process. The purified enzyme preparation was apparently homogeneous as examined by the polyacrylamide gel electrophoresis, analytical gel-permeation chromatography, and analytical ultracentrifugation. The final enzyme preparation showed a purification of 803-fold from the culture medium with a specific activity of 578 U/mg of protein. Fluorescence spectroscopic analyses showed multiple unfolding steps in the urea denaturation process of the homodimeric recombinant enzyme. Extensive conformational change of the enzyme in urea was detected by the analytical ultracentrifugation and the size-exclusive chromatography. The quaternary structure of the enzyme is quite stable. No indication of dissociation was observed after extensive tertiary structural changes.
Chen
Yu-Hou
YH
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC.
Chang
Tsu-Chung
TC
Chang
Gu-Gang
GG
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000469:Alkaline Phosphatase; D003001:Cloning, Molecular; D004795:Enzyme Stability; D058851:GPI-Linked Proteins; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D058851:GPI-Linked Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental