Overexpression and purification of scytovirin, a potent, novel anti-HIV protein from the cultured cyanobacterium Scytonema varium.
Scytovirin (SVN) is a novel anti-human immunodeficiency virus (HIV) protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds. A synthetic gene that encodes scytovirin was constructed, and expressed in Escherichia coli, with thioredoxin (TRX) fused to its N-terminus (TRX-SVN). Most of the expressed protein was in soluble form, which was purified by a polyhistidine tag affinity purification step. SVN was then cleaved from TRX with enterokinase and separated from the TRX partner by C18 reversed-phase HPLC. This production method has proven superior to earlier synthetic attempts and recombinant procedures using a standard expression system. The current system resulted in yields of 5-10mg/L of purified SVN for structural studies and for preclinical development of SVN as a topical microbicide for HIV prophylaxis.
Xiong
Changyun
C
Molecular Targets Development Program, National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD, USA.
O'Keefe
Barry R
BR
Botos
Istvan
I
Wlodawer
Alexander
A
McMahon
James B
JB
D019380:Anti-HIV Agents / Q000302:isolation & purification* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli; D015658:HIV Infections / Q000517:prevention & control*; D037102:Lectins; D008565:Membrane Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D037102:Lectins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C474342:scytovirin protein, S varium