Journal article
Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system
Applied biochemistry and biotechnology, Vol.101(3), pp.197-210
06/2002
DOI: 10.1385/ABAB:101:3:197
PMID: 12109816
Abstract
Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that V
max and K
m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V
max and K
m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man2–8 GlcNAc2 and FucMan3 or 4GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells.
Details
- Title: Subtitle
- Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system
- Creators
- Fuming Zhang - Department of Chemistry and Division of Medicinal and Natural Products Chemistry University of Iowa 52242 Iowa City IADavid Murhammer - Department of Chemical and Biochemical Engineering University of Iowa 52242 Iowa City IARobert Linhardt - Department of Chemistry and Division of Medicinal and Natural Products Chemistry University of Iowa 52242 Iowa City IA
- Resource Type
- Journal article
- Publication Details
- Applied biochemistry and biotechnology, Vol.101(3), pp.197-210
- Publisher
- Humana Press; Totowa
- DOI
- 10.1385/ABAB:101:3:197
- PMID
- 12109816
- ISSN
- 0273-2289
- eISSN
- 1559-0291
- Language
- English
- Date published
- 06/2002
- Academic Unit
- Chemical and Biochemical Engineering
- Record Identifier
- 9984004191102771
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