Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity

详细信息    查看全文

• 作者：Hongling Yuan ; Quan Du ; Matthew B. Sturm ; Vern L. Schramm
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：July 28, 2015
• 年：2015
• 卷：54
• 期：29
• 页码：4565-4574
• 全文大小：424K
• ISSN：1520-4995

文摘

Saporin L3 from Saponaria officinalis (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 Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >10³ 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 Pichia pastoris when it is linked to the protein export system for the yeast 伪-mating factor. DNA encoding saporin L3 was cloned into a pPICZ伪B expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the 伪-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the 伪-mating factor yielded truncated saporin L3 (amino acids 22鈥?80), 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.