title: The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.
abstract: The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only ∼54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 °C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at ∼5 °C and 1 M NaCl. The MLuc2 adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations.
authors.affiliation: Photobiology Lab, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russia; Siberian Federal University, Krasnoyarsk, Russia.
authors.forename: Marina D
authors.initials: MD
authors.lastname: Larionova
authors.affiliation: Photobiology Lab, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russia; Siberian Federal University, Krasnoyarsk, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Markova
authors.affiliation: Photobiology Lab, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russia; Siberian Federal University, Krasnoyarsk, Russia. Electronic address: eugene.vysotski@gmail.com.
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D033342:Copepoda / Q000201:enzymology*; D004795:Enzyme Stability; D006358:Hot Temperature; D007313:Insecta / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D049449:Luminescence; D008163:Luminescent Measurements; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D007527:Isoenzymes; D008156:Luciferases
keywords: Bioluminescence; Bioluminescent reporter; Coelenterazine; Molecular adaptation; Psychrophilic enzyme