Identification of new dominant-negative mutants of anthrax protective antigen using directed evolution.

Abstract	The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment.
Authors	Gaobing Wu, Chunfang Feng, Sha Cao, Aizhen Guo, Ziduo Liu
Journal	Applied biochemistry and biotechnology (Appl Biochem Biotechnol) Vol. 168 Issue 5 Pg. 1302-10 (Nov 2012) ISSN: 1559-0291 [Electronic] United States
PMID	22948605 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Antigens, Bacterial Bacterial Toxins Mutant Proteins anthrax toxin
Topics	Animals Anthrax (genetics, immunology, microbiology) Antigens, Bacterial (chemistry, genetics, immunology, isolation & purification) Bacillus anthracis (genetics, immunology, pathogenicity) Bacterial Toxins (chemistry, genetics, immunology, isolation & purification) CHO Cells Cricetinae Directed Molecular Evolution Humans Mice Mutant Proteins (genetics, immunology, isolation & purification) Mutation Protein Structure, Tertiary

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.

Realize the full power of the drug-disease research graph!