Abstract
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.
Highlights
The resolving power of modern immunoassays depends on both specificity and affinity of antibodies as well as the efficiency of reporter molecules in converting antibody-analyte complex concentration into a detectable signal
We report for the first time the construction of two variants of a hybrid protein consisting of the smallest isoform of Metridia luciferase (MLuc7) [19] as a bioluminescent reporter and a murine single-chain variable fragment mini-antibody to the glycoprotein E of tick-borne encephalitis virus (TBEV) [35], being a surface envelope protein that plays a key role in virus penetration into host cells
To evaluate the ability of the smallest MLuc7 luciferase to retain its functional properties as a part of hybrid protein and to determine the best options for a design of future reporter constructs, two variants of a hybrid protein (Figure 1a) consisting of the MLuc7 as a bioluminescent reporter and a murine single-chain variable fragment antibody against the glycoprotein E of tick-borne encephalitis virus (TBEV) [35] were obtained
Summary
The resolving power of modern immunoassays depends on both specificity and affinity of antibodies as well as the efficiency of reporter molecules in converting antibody-analyte complex concentration into a detectable signal For this purpose, antibodies or analytes are frequently linked to luciferases or Ca2+-regulated photoproteins [1,2,3,4]. Antibodies or analytes are frequently linked to luciferases or Ca2+-regulated photoproteins [1,2,3,4] These proteins catalyze various bioluminescent reactions and are responsible for the bioluminescence of various marine and terrestrial organisms [5]. To achieve covalent linkage between antibody or analyte and a protein reporter, the use of cross-linking chemicals is the norm This often yields a mixture of conjugated molecules with different affinities and decreases, or even annuls, the activity of signal enzyme, diminishing detection power. These are primarily their invariable ratio of recognizing domain/reporter protein and their constant orientation relative to each other
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