Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity YuanHongling DuQuan SturmMatthew B. SchrammVern L. 2015 Saporin L3 from <i>Saponaria officinalis</i> (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple <i>Escherichia coli</i> vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in <i>E. coli</i> nonexpression strains. Saporin L3 is >10<sup>3</sup> times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in <i>Pichia pastoris</i> when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in <i>P. pastoris</i> under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the <i>P. pastoris</i> cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22–280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.