Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development
| Autor: | Karyn McFadden, Karen M. Watson, Tara Pagliei, Irwin Chaiken, Ryan Kuss, Diana M. Colleluori, Deborah Tien, Robert W. Buckheit, Feirong Kang, Timothy McCormick, Joseph W. Romano |
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| Rok vydání: | 2004 |
| Předmět: |
Anti-HIV Agents
Blotting Western Genetic Vectors Gene Expression Enzyme-Linked Immunosorbent Assay HIV Envelope Protein gp120 medicine.disease_cause Inclusion bodies law.invention Inclusion Bodies Viral Inhibitory Concentration 50 Bacterial Proteins law Sequence Analysis Protein Microbicide Enzyme Stability medicine Escherichia coli Animals Humans Cloning Molecular Polyacrylamide gel electrophoresis Chromatography High Pressure Liquid biology Isoelectric focusing Molecular biology Recombinant Proteins Cyanovirin-N Cell culture Vagina biology.protein Recombinant DNA Chromatography Gel Electrophoresis Polyacrylamide Gel Female Carrier Proteins Biotechnology HeLa Cells |
| Zdroj: | Protein expression and purification. 39(2) |
| ISSN: | 1046-5928 |
| Popis: | Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development. |
| Databáze: | OpenAIRE |
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