Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris.
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.
Rajamohan
F
F
Biotherapy Program, Hughes Institute, Roseville, Minnesota 55113, USA. frajamohan@ih.org
Doumbia
S O
SO
Engstrom
C R
CR
Pendergras
S L
SL
Maher
D L
DL
Uckun
F M
FM
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
D019380:Anti-HIV Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein