Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development.
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.
Colleluori
Diana M
DM
Biosyn, Incorporated, 1800 Byberry Road, Building 13, Huntingdon Valley, PA 19006, USA. colleluori@biosyn-inc.com
Tien
Deborah
D
Kang
Feirong
F
Pagliei
Tara
T
Kuss
Ryan
R
McCormick
Timothy
T
Watson
Karen
K
McFadden
Karyn
K
Chaiken
Irwin
I
Buckheit
Robert W
RW
Romano
Joseph W
JW
D000818:Animals; D019380:Anti-HIV Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007181:Inclusion Bodies, Viral / Q000378:metabolism; D020128:Inhibitory Concentration 50; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014621:Vagina / Q000187:drug effects* / Q000382:microbiology
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; C107733:cyanovirin N