1. Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development
- Author
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Karyn McFadden, Karen M. Watson, Tara Pagliei, Irwin Chaiken, Ryan Kuss, Diana M. Colleluori, Deborah Tien, Robert W. Buckheit, Feirong Kang, Timothy McCormick, and Joseph W. Romano
- Subjects
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 - Abstract
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.
- Published
- 2004