pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens.
We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity. Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus. As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins. All three recombinant allergens were readily detected in nitrocellulose-blotted E. coli extracts by the Bp36/51-specific moAb 4A6. We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting. A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients. Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera. We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g. IgE), even when they occur in minute concentrations.
Pandjaitan
B
B
Institute of Medical and Chemical Laboratory Diagnostics, Vienna General Hospital, University of Vienna, Vienna, Austria.
Eibensteiner
P B
PB
Vrtala
S
S
Hayek
B
B
Grote
M
M
Reichelt
R
R
Rumpold
H
H
Valenta
R
R
Spitzauer
S
S
D000485:Allergens / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003285:Contractile Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D007073:Immunoglobulin E / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics* / Q000276:immunology; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011058:Pollen / Q000235:genetics / Q000276:immunology; D051304:Profilins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012995:Solubility
D000485:Allergens; D000911:Antibodies, Monoclonal; D003285:Contractile Proteins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010940:Plant Proteins; D051304:Profilins; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E