Тип публикации: статья из журнала
Год издания: 2020
Идентификатор DOI: 10.3390/ijms21144971
Ключевые слова: bioluminescence, coelenterazine, copepod luciferase, single-chain antibody, immunoassay, tick-borne encephalitis virus, tick-borne encephalitis virushistidine, 645-35-2, 7006-35-1, 71-00-1, single chain fragment variable antibody, 334577-34-3, 334577-38-7
Аннотация: 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 copepodMetridia longa. In this sПоказать полностьюtudy, 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 (K-D= 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 (K-D= 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 ofM. longaluciferase 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.
Журнал: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Выпуск журнала: Vol. 21, Is. 14
Номера страниц: 4971
ISSN журнала: 14220067
Место издания: BASEL
Издатель: MDPI