Cyanovirin-N (CV-N) is under development as a topical (vaginal or rectal) microbicide to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of the protein is all urgent priority. We observed that N-glycosylation of CV-N in yeast eliminated the anti-HIV activity, and that dimeric forms and aggregates of CV-N occurred under certain conditions, potentially complicating the efficient large-scale manufacture of pure monomeric CV-N. We therefore expressed and tested CV-N homologs in which the Asn residue at position 30 was replaced with Ala, Gin, or Val, and/or the Pro at position 51 was replaced by Gly to eliminate potential conformational heterogeneity. All homologs exhibited anti-HIV activity comparable to wild-type CV-N and the Pro51Gly homologs were significantly more stable proteins. These glycosylation-resistant, functional cyanovirins should he amenable to large-scale production either in bacteria or in eukaryotic hosts. (C) 2002 Elsevier Science (USA). All rights reserved.