title: Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris.
abstract: Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of homogeneously pure active PAP without batch-to-batch variation from its natural resource. Here, we report the expression of mature PAP (residues 23 to 284) with a C-terminal hexahistidine tag in the methylotrophic yeast Pichia pastoris, as a secreted soluble protein. The final yield of the secreted PAP is greater than 10 mg/L culture in shaker flasks. The secreted recombinant protein is not toxic to the yeast cells and has an apparent molecular mass of 33-kDa on SDS-PAGE gels. The in vitro enzymatic activity and cellular anti-HIV activity of recombinant PAP were of the same magnitude as those of the native PAP purified from P. americana. To our knowledge, this is the first large-scale expression and purification of soluble and biologically active recombinant mature PAP from yeast.
authors.affiliation: Biotherapy Program, Hughes Institute, Roseville, Minnesota 55113, USA. frajamohan@ih.org
authors.forename: F
authors.initials: F
authors.lastname: Rajamohan
authors.forename: S O
authors.initials: SO
authors.lastname: Doumbia
authors.forename: C R
authors.initials: CR
authors.lastname: Engstrom
authors.forename: S L
authors.initials: SL
authors.lastname: Pendergras
authors.forename: D L
authors.initials: DL
authors.lastname: Maher
authors.forename: F M
authors.initials: FM
authors.lastname: Uckun
mesh_terms: D019380:Anti-HIV Agents / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D005822:Genetic Vectors; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000821:virology; D009699:N-Glycosyl Hydrolases; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D014170:Transformation, Genetic; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein