Generation of monoclonal antibodies against Escherichia coli DsbA.
Disulfide oxidoreductase A (also known as disulfide bond formation protein A, or DsbA) is produced in Escherichia coli (E. coli) periplasm, which plays an important role in correct formulation of disulfide bonds during protein exportation in vivo. DsbA prokaryotic expression vectors are designed for periplasmic co-expression of recombinant proteins with an improving secretion. In the present study, the first domain of human CD226 (CD226D1) was expressed as a His-tagged fusion protein with DsbA and purified by Ni-NTA resin. Three monoclonal antibodies (MAbs) against DsbA were raised by using the recombinant fusion protein as immunogen. We have demonstrated that one of them can detect DsbA via Western blotting, in addition to its ability to immunoprecipitate DsbA fusion protein. Furthermore, anti-DsbA MAb can be employed to prepare antibody-coupled affinity column for purification of DsbA fusion protein from E. coli lysate.
Zhang
Yuan
Y
Department of Immunology, Fourth Military Medical University, Xi'an-Shaanxi Province, PR China.
Zhuang
Ran
R
Xu
Zhu-Wei
ZW
Jin
Bo-Quan
BQ
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000502:physiology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004220:Disulfides; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D019704:Protein Disulfide-Isomerases