1. A General Method for Rapid Purification of Soluble Versions of Glycosyhosphatidylinositol-Anchored Proteins Expressed in Insect Cells: An Application for Human Tissue-Nonspecific Alkaline Phosphatase1.
- Author
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Oda, Kimimitsu, Amaya, Yoshihiro, Fukushi-Irie, Mariko, Kinameri, Yasuko, Ohsuye, Kazuhiro, Kubota, Ichiro, Fujimura, Shinichi, and Kobayashi, Jun
- Subjects
PROTEINS ,ALKALINE phosphatase ,PHOSPHATASES ,ZINC enzymes ,OSTEOSARCOMA ,BONE cancer - Abstract
A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. I mm u nob lotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: iV-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyaery 1 amide gel, indicating that the size difference between the two enzymes is ascribed to iV-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluore-scence studies. [ABSTRACT FROM AUTHOR]
- Published
- 1999
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