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
Source: PubMed


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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    • "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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    • "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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