Recombinant Hybrid Proteins as Biospecific Reporters for Bioluminescent Microassay

Abstract

Light-emitting proteins, luciferases and photoproteins, are widely used as reporters in high sensitive binding assays in vitro and in vivo. The combination of luciferase and biospecific molecule with affinity to a certain target (immunoglobulin, oligonucleotide, hapten, etc.) promotes specificity of the assay. The molecules of the kind (biospecific bioluminescent labels) are usually obtained by chemical synthesis using different commercial reagents. However, this approach cannot provide homogeneity of the product; it causes the loss (sometimes essential) of specific activity of the initial molecules. As a rule chemical binding requires a thorough verification of the reaction conditions (balance of reagents, pH and temperature of the media, reaction duration, etc.) that would decrease a negative impact of the reagents and improve the yield of conjugates. Another way to obtain combination of the kind is the creation of a hybrid protein by using genetic engineering approach. In this case a genetic construction containing, in one frame, genes coding biospecific and reporter proteins as well as sequences, coding a linker between them and sometimes auxiliary polypeptides, is created. Gene fusion approach provides easy bacterial production, hybrids homogeneity, easy purification using auxiliary polypeptides etc. The paper describes several hybrids “luciferase–biospecific polypeptide” designed in the photobiology lab of the Institute of Biophysics SB RAS, their construction, production and application in microassay as biospecific bioluminescent probes