The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins

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.

Coelenterazine-Dependent Luciferases: Properties and Application

“…Люциферазы, в отличие от фотопротеинов, чувствительны к химическим модификациям и при попытке синтезировать специфические репортеры теряют значительную часть активности. Поэтому для анализа in vitro чаще всего получают гибридные варианты люцифераз, генетически-слитых с адресными полипептидами [6,[44][45][46][47][48][49], или конструируют варианты, содержащие уникальные аминокислоты, обеспечивающие сайт-направленную химическую модификацию, которая практически не затрагивает биолюминесцентную функцию белка [50,51].…”

Antibodies as Biosensors’ Key Components: State-of-the-Art in Russia 2020–2021

“…These are primarily their invariable ratio of the recognition and reporter components and their constant orientation with respect to each other. A test system based on the sandwich-type bioluminescent immunoassay has been developed [24]. This work constructed hybrid bifunctional proteins consisting of the recognition domain-a murine single-chain variable fragment mini-antibody (scFv 14D5a) specific to glycoprotein E of the tick-borne encephalitis virus.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…As a sensory component of hybrids, various biospecific polypeptides were applied—antibodies or their fragments [ 111 , 112 , 113 , 114 ], antigens [ 115 , 116 , 117 , 118 ], polypeptides with special binding ability [ 119 , 120 , 121 , 122 ], GFP and its color variants [ 123 , 124 , 125 , 126 , 127 , 128 , 129 ], etc. Based on the phenomenon of assembling split luciferase derivatives, a whole spectrum of homogeneous methods has been developed [ 125 , 126 , 127 , 128 , 129 ] and applied for studying protein–protein/ligand interaction [ 130 , 131 , 132 , 133 ], protein activity [ 134 , 135 ], and protein folding [ 136 , 137 ].…”