Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

Abstract

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (lambda(max)=480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.

Purification and characterization of a novel thermostable luciferase from Benthosema pterotum

“…6a) and a relative high residual activity at pH 12 (ffi40% of the maximum). This profile appears similar to those of copepod Gaussia princeps luciferase, squid Watasenia scintillans and copepod Metridia luciferase [28][29][30]. In the case of some luciferase, photoproteins can be inactive at some pH values, without resulting in permanent inactivation.…”

The smallest natural high-active luciferase: Cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa

“…This recombinant MLuc7 has $80-fold greater specific bioluminescence activity than monomeric MLuc7 purified from E. coli (Table 1), i.e. the difference of activities is higher than that between similar samples of MLuc164 isoform [17]. Some other bioluminescence properties of these MLuc7 samples (pH and temperature profiles of light intensities, thermostability) also differed (Fig.…”

“…The MLuc7 coding sequence with native signal peptide was amplified using specific primers: forward 5 0 -GACGGATCCATGGATATCAAATT-TATTTT-3 0 with BamHI site (underlined) and two overlapping reverse primers: first 5 0 -TGATGATGACCTTGAAAGTACA AGTTCTCACGATCTCCAGCAAGAC-3 0 and second 5 0 -TACTC-GAGTCATTAGTGATGGTGATGGTGATGATGACCTTGAAAG-3 0 with XhoI site, using two-step PCR as described for MLuc164 [17]. The oligonucleotide primers were designed to introduce the C-terminal His6-tag followed by a TEV-specific protease site after MLuc7 coding sequence.…”

“…However, this E. coli compartment is not always efficient for proper disulfide bond formation in proteins having numerous SAS bonds [16]. Expression in insect cells with the use of baculovirus expression system lacks these shortcomings; the luciferase MLuc164 from Metridia longa, for example, expressed in the insect cell system is a monomeric protein with a 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli [17].…”

Bioluminescent monitoring enables observation of intracellular events in real time without cell and tissue destruction

“…longa cодеp-жат на N-конце cигнальную поcледовательноcть из 17 аминокиcлотныx оcтатков, котоpая обеc-печивает эффективную cекpецию pекомбинант-ныx люцифеpаз пpи иx экcпpеccии в клеткаx млекопитающиx и наcекомыx [2,4,7]. Cpавнение поcледовательноcтей изофоpм выявляет ваpиа-бельный N-конец, pазмеpом около 1/3 поcле-довательноcти наибольшей изофоpмы MLuc164, и конcеpвативный C-конец, отвечающий за ка-талитичеcкую активноcть.…”