Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli
| Autor: | David E. Lanar, K. Fegeding, Lisa A. Ware, V. L. Miller, M. A. Vassell, John B. Sacci, Svetlana Kitov, Nelly Kolodny, P De La Vega |
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| Rok vydání: | 2001 |
| Předmět: |
Molecular Sequence Data
Plasmodium falciparum Protozoan Proteins medicine.disease_cause Chromatography Affinity Cell Line Sepharose chemistry.chemical_compound Affinity chromatography Protein A/G medicine Escherichia coli Animals Humans Amino Acid Sequence Cloning Molecular Chromatography biology Chemistry General Chemistry biology.organism_classification Chromatography Ion Exchange Recombinant Proteins Circumsporozoite protein Biochemistry Cell culture biology.protein Agarose Electrophoresis Polyacrylamide Gel |
| Zdroj: | Journal of chromatography. B, Biomedical sciences and applications. 762(1) |
| ISSN: | 1387-2273 |
| Popis: | The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 microg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml. |
| Databáze: | OpenAIRE |
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