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.