A baculovirus-expressed fusion protein containing the antibody-binding domain of protein A and insect luciferase.
A fusion construct encoding two antibody-binding sites of protein A from Staphylococcus aureus and click beetle, Pyrophorus plagiophthalamus, luciferase (LucGR) was designed and expressed using the baculovirus system. The construct was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in the insect Spodoperta frugiperda cell line during viral infection. The properties of the resultant chimeric protein product, protA-LucGR, were studied both in vivo and in vitro by using i) luminometry, ii) immunoblot analysis, iii) immunoprecipitation, iv) metabolic labeling procedures and v) luminescent immunoassays. Together, the results clearly demonstrate that the light-emitting properties of the fused luciferase construct remain intact. Further, the antibody-binding domain of protein A retains its activity as it binds to both rabbit and goat as well as human immunoglobulins. Due to the dual biological function of this fusion protein, it should provide a potential reagent within the field of molecular biology and diagnostics.
Oker-Blom
C
C
Abo Akademi University, Turku, Finland.
Suomalainen
A M
AM
Akerman
K
K
Qi
Z
Z
Lindqvist
C
C
Kuusisto
A
A
Karp
M
M
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D001709:Biotechnology; D015153:Blotting, Western; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005821:Genetic Techniques; D005822:Genetic Vectors; D007915:Lepidoptera / Q000235:genetics; D008156:Luciferases / Q000235:genetics* / Q000276:immunology; D008163:Luminescent Measurements; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008156:Luciferases