Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris

Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca ame ricana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potenti al anti-HIV agent. However, the clinical use of native PAP is limited due t o 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, a s a secreted soluble protein. The final yield of the secreted PAP is greate r than 10 mg/L culture in shaker flasks. The secreted recombinant protein i s 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 ac tivity 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 recomb inant mature PAP from yeast. (C) 2000 Academic Press.