bi5b00405_si_001.pdf (148.93 kB)
Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity
journal contribution
posted on 2015-07-28, 00:00 authored by Hongling Yuan, Quan Du, Matthew B. Sturm, Vern L. SchrammSaporin 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 α-mating factor. DNA encoding saporin L3 was cloned
into a pPICZαB 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
α-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 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.
History
Usage metrics
Categories
Keywords
Pichia pastorissaporin L 3eukaryotic rRNAcoli nonexpression strainsplasmid maintenanceDNA encoding saporin L 3pPICZ αB expression vectorprotein export systemSaporin L 3fusion proteinSaponaria officinalissaporin L 3 expressionmethanol inductionGAGA tetraloopSoapwort Saporin L 3 Expressionpastoris cellsalcohol dehydrogenase AOX 1 promoterculture mediumDepurination sitesRNA deadenylationRNA Substrate SpecificitySaporin L 3Multiple Escherichia coli vector designs32 kDarelease adeninesaporin isoform
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC