Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts.
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 an 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 glycosylation-susceptible Asn residue at position 30 was replaced with Ala, Gln, 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 be amenable to large-scale production either in bacteria or in eukaryotic hosts.
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D019380:Anti-HIV Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells; D006031:Glycosylation; D015497:HIV-1 / Q000187:drug effects; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000235:genetics*; D011387:Prokaryotic Cells; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics; D014170:Transformation, Genetic
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C107733:cyanovirin N; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase