Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.
Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.
Zhang
F
F
Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City 52242, USA.
Wolff
M W
MW
Williams
D
D
Busch
K
K
Lang
S C
SC
Murhammer
D W
DW
Linhardt
R J
RJ
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D015870:Gene Expression / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007239:Infections / Q000821:virology; D007304:Insect Viruses / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000166:cytology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D000469:Alkaline Phosphatase