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

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Protein Expression and Purification (Impact Factor: 1.7). 07/2008; 61(2):142-8. DOI: 10.1016/j.pep.2008.05.013
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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.

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Available from: Eugene S Vysotski, Oct 03, 2015
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    • "These VHH molecules were previously generated functionally in E. coli. However, the use of insect host cells to produce VHHs would provide further advantages, such as the possibility to make posttranslational modifications, including the formation of disulphide bonds [36]. In this case, llama VHHs, which have an extended CDR3 that is often stabilized by an additional disulphide bond with a cysteine residue in CDR2 [1,37], would be more efficiently produced. "
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    ABSTRACT: Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule.
    BMC Biotechnology 09/2012; 12(1):59. DOI:10.1186/1472-6750-12-59 · 2.03 Impact Factor
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    • "The light emission spectra of ML164, ML164M3, and ML164M4 reveal peaks in the range of 496–502 nm with a full width at halfmaximum intensity of 80 nm (Fig. 2C). These values are very close to those which were published earlier for different preparations of M. longa luciferases [8] [22] [24]. The M. longa luciferase like other proteins containing no chromophores, has no fluorescence in the visible range. "
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    ABSTRACT: The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.
    Biochemical and Biophysical Research Communications 11/2011; 417(1):98-103. DOI:10.1016/j.bbrc.2011.11.063 · 2.30 Impact Factor
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    • "Heterologous reporter proteins, such as secreted alkaline phosphatase, have been used to examine the regulation of secreted protein synthesis, because these reporters can be detected using standardized, rapid assays, without need for Western blot. For our reporter, we decided to use the luciferase of the marine copepod Metridia longa, which possesses an endoplasmic reticulum targeting signal sequence [32]. We used fusion PCR to replace the native Metridia longa luciferase leader peptide, MDIKVVFTLVFSALVQA, with the envelope leader peptides of (1) AA01; (2) position 12 histidine to glutamine mutant of AA01; (3) AC01. "
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    ABSTRACT: Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.
    PLoS ONE 08/2011; 6(8):e23673. DOI:10.1371/journal.pone.0023673 · 3.23 Impact Factor
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