Journal article
Phosphorylatable and epitope-tagged human erythropoietins: Utility and purification of native baculovirus-derived forms
Protein expression and purification, Vol.3(6), pp.461-469
1992
DOI: 10.1016/1046-5928(92)90063-3
PMID: 1283094
Abstract
The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for
in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high
in vitro activity (⩾500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOS, forms modified at the amino terminus (Ala
1) fully retained receptor binding and
in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys
161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of
32
32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.
Details
- Title: Subtitle
- Phosphorylatable and epitope-tagged human erythropoietins: Utility and purification of native baculovirus-derived forms
- Creators
- Dawn E Quelle - Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USAKevin J Lynch - Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USARebecca E Burkert-Smith - Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USAStefan Weiss - GIBCOBRL Life Technologies, Grand Island, New York 14072, USAWilliam Whitford - GIBCOBRL Life Technologies, Grand Island, New York 14072, USADon M Wojchowski
- Resource Type
- Journal article
- Publication Details
- Protein expression and purification, Vol.3(6), pp.461-469
- Publisher
- Elsevier Inc
- DOI
- 10.1016/1046-5928(92)90063-3
- PMID
- 1283094
- ISSN
- 1046-5928
- eISSN
- 1096-0279
- Language
- English
- Date published
- 1992
- Academic Unit
- Pathology; Neuroscience and Pharmacology
- Record Identifier
- 9984040484302771
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