Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity.
Fungal ribotoxins are a family of extracellular ribonucleases which inhibit protein biosynthesis by inactivating the ribosomes. This inactivation results in the induction of cell death by apoptosis. Ribotoxins show antitumoral properties based on their ability to cross the membrane of some transformed cells. Unfortunately, they also show an unspecific cytotoxicity which has greatly impaired their potential clinical uses. alpha-Sarcin, produced by Aspergillus giganteus, is the best-characterized ribotoxin. Asp f 1, another ribotoxin produced by A. fumigatus, is indeed one of its major allergens. In this work, the Lactococcus lactis MG1363 strain has been engineered to produce and secrete not only wild-type Asp f 1 and alpha-sarcin but also three different mutants with reduced cytotoxicity and/or IgE-binding affinity. The proteins were secreted in native and active form when the extracellular medium employed was buffered at pH values around 8.0. Strains producing the wild-type natural alpha-sarcin were proved to be innocuous when administered intragastrically to mice for a period of 14 days. Overall, the results presented are discussed in terms of its potential application as a vehicle of oral delivery of hypoallergenic variants as well as a starting point to approach the design of strategies to accomplish the safe delivery of these proteins as antitumoral agents.
Alvarez-García
Elisa
E
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain.
Alegre-Cebollada
Jorge
J
Batanero
Eva
E
Monedero
Vicente
V
Pérez-Martínez
Gaspar
G
García-Fernández
Rosa
R
Gavilanes
Jose G
JG
del Pozo
Alvaro Martínez
AM
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D007073:Immunoglobulin E / Q000378:metabolism*; D007421:Intestine, Small / Q000382:microbiology; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009183:Mycotoxins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding
D005656:Fungal Proteins; D009183:Mycotoxins; C014801:alpha-sarcin; D007073:Immunoglobulin E; D004722:Endoribonucleases