A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: An application for human tissue-nonspecific alkaline phosphatase

A soluble form of tissue-nonspecific alkaline phosphatase was purified to a pparent homogeneity from the culture media of Sf9 cells which had been infe cted with recombinant baculoviruses encoding human tissue-nonspecific alkal ine phosphatase (TNSALP). To facilitate purification, an oligonucleotide co nsisting of 6 tandem codons for histidine and a stop codon was engineered i nto the TNSALP cDNA. The molecular mass of the enzyme purified through a ni ckel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (S DS)-polyacrylamide gel electrophoresis, That of the native enzyme was 90 kD a as estimated by gel filtration, indicating that the purified soluble TNSA LP is dimeric, The enzyme was used for production of antibodies specific fo r human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, u pon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human ori gin and the soluble enzyme of insect origin migrated to the same position o n SDS-polyacrylamide gel, indicating that the size difference between the t wo enzymes is ascribed to N-linked oligosaccharides. The antibodies prepare d against the purified TNSALP were found to be useful also for immunoprecip itation and immunofluorescence studies.