Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells.
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.
Stepanyuk
Galina A
GA
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Xu
Hao
H
Wu
Chia-Kuei
CK
Markova
Svetlana V
SV
Lee
John
J
Vysotski
Eugene S
ES
Wang
Bi-Cheng
BC
D000818:Animals; D003001:Cloning, Molecular; D003445:Crustacea / Q000201:enzymology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D049449:Luminescence; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008156:Luciferases