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Protein Expr Purif. 2005 Feb;39(2):229-36.

Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development.

Author information

1
Biosyn, Incorporated, 1800 Byberry Road, Building 13, Huntingdon Valley, PA 19006, USA. colleluori@biosyn-inc.com

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.

PMID:
15642474
DOI:
10.1016/j.pep.2004.10.009
[Indexed for MEDLINE]

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