Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays.
The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the beta subunit of luciferase. Only the first fusion protein encoding the entire beta subunit was able to form an enzymatically active luciferase complex when expressed together with the alpha subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.
Lindbladh
C
C
Department of Pure and Applied Biochemistry, University of Lund, Sweden.
Mosbach
K
K
Bülow
L
L
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007118:Immunoassay; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014733:Vibrio / Q000201:enzymology
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008156:Luciferases