Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells.
The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.
Rodríguez-Díaz
Jesús
J
Department of Microbiology, School of Medicine and Hospital Clínico Universitario, University of Valencia, 46010 Valencia, Spain.
López-Andújar
Pilar
P
García-Díaz
Ana
A
Cuenca
Javier
J
Montava
Rebeca
R
Buesa
Javier
J
D000818:Animals; D002478:Cells, Cultured; D003967:Diarrhea / Q000139:chemically induced; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006031:Glycosylation; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D011993:Recombinant Fusion Proteins; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; C033223:polyhistidine; D006639:Histidine