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WO2017209700A1 - Fusion bacterial luciferase gene, reporter vector, and assay kit - Google Patents

Fusion bacterial luciferase gene, reporter vector, and assay kit Download PDF

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Publication number
WO2017209700A1
WO2017209700A1 PCT/TH2016/000051 TH2016000051W WO2017209700A1 WO 2017209700 A1 WO2017209700 A1 WO 2017209700A1 TH 2016000051 W TH2016000051 W TH 2016000051W WO 2017209700 A1 WO2017209700 A1 WO 2017209700A1
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fusion
bacterial luciferase
gene
cell
luciferase
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Pimchai CHAIYEN
Ruchanok TINIKUL
Jittima PHONBUPPHA
Paweena CHOOCHUAY
Autchara WONGSATHEP
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Mahidol University
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Mahidol University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14003—Alkanal monooxygenase FMN (1.14.14.3), i.e. bacterial-luciferase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • This invention relates to a fusion bacterial luciferase gene, reporter vector, and assay kit wherein the bacterial luciferase gene, specifically a codon optimized luxA and hixB fusion bacterial luciferase, is incorporated into an expression vector and is transfected into a cell or a group of cells of interest.
  • a promoter or regulation sequence of interest can be further incorporated to the vector to investigate its effect on certain gene expression or regulation, or its response to external compound via a bacterial luciferase activity by which a complimentary set of assay kit has been developed to enable the reaction and measurement of said bacterial luciferase expression activity inside the cell sample.
  • the fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention can be further developed into a high-throughput drug screening system.
  • Luciferases have been widely used in bioluminescence- based detection technology. From 2010 survey data, nearly 2,000 assays are listed in PubChem database. Thus, about 21% of the available detection methods are relying on bioluminescence- based detection ( Thorne et al. , 2010) . Advantages of bioluminescence- based detection over fluorescence- based systems include no requirement of exogenous excitation light which allows signal generation with low background and eliminates photo-bleaching side effects (Waidman et al., 2011). Thus, luminescence detection typically gives higher sensitivity than fluorescence- based methods. The bioluminescence system is also suitable for measurement of dynamic changes due to its relatively short protein half- life (Allen et al. , 2007) . Therefore, the use of bioluminescence- based bioassays has increased continuously.
  • Bacterial luciferase produces blue- green light ( max 490 nm) by oxidation of reduced Flavin mononucleotide (FMN) and long chain aldehyde (RCHO) using oxygen (0 2 )
  • bioluminescence quantum yield of bacterial luciferase is about 10-16% (Shimomura, 2006; Lei et al., 2004).
  • Bacterial luciferase is encoded by adjacent duplicated luxA and luxB genes in the lux operon.
  • luxA and luxB genes in the lux operon.
  • a long chain aldehyde is synthesized by a fatty acid reductase multi- enzymes complex, the gene products of luxC, luxD and luxE in the same lux operon.
  • LuxD is encoded for a transferase catalysing the hydrolysis of fatty acyl group to fatty acid.
  • luxC and luxE the gene products of luxC and luxE, consequently catalyse reduction of fatty acid to aldehyde to supply for luciferase reaction.
  • the NADtLFMN oxidoreductase encoded by the luxG gene in the lux operon is responsible for generating majority of reduced FMN to supply the luminescence reaction (Nijvipakul et al., 2008).
  • Bacterial luciferase is a heterodimeric protein and is composed of a- and ⁇ -subunits with approximately 77 kDa. These two luciferase subunits share about 30% sequence identity with molecular mass about 40-42 kDa for a-subunit and 36-37 kDa for ⁇ -subunit (Szittner et al., 1990).
  • luciferase Several properties allow bacterial luciferase to be a good reporter enzyme in bioluminescence- based detection technology. It is a very sensitive sensor as only about 10 pg of luciferase (Meighen et al., 1991) or 10 5 luciferase molecules can be detected (Olsson et al., 1989) by a conventional luminometer method.
  • Luminescence detection is advantageous for the fact that there is usually no endogenous background in non-luminous organisms as compared to the detection by green florescence protein (Waidmann et al., 2011). Luciferase assay is generally fast and requires only a few seconds to process. In addition, light intensity can be correlated well with the amount of luciferase enzyme and mRNA transcripts over a wide range. This is because the luciferase assay reaction undergoes only a single catalytic turnover.
  • the compounds are much cheaper than luciferin and ATP used in firefly luciferase or colelenterazine used in renilla luciferase (Alam et al., 1990).
  • aldehyde substrate itself can also rapidly traverse the cell membrane (Meighen, 1993).
  • the bacterial luciferase system possesses a distinct advantage over firefly and renilla luciferase systems in that it has a complete set of genes for all substrates synthesis in the same operon, which also allows the system to be self-luminous without cell destruction and exogenous substrate adding if the whole set of genes is expressed.
  • Bacterial luciferase has been used as a reporter or sensor for detection of various compounds, cell viability and distribution, and in vitro and in vivo metabolic function (Meighen, 1991; Ulitzur,
  • V. haveyi (Vh) fusion luciferase expressed at 37°C displayed in vivo bioluminescence level in E. coli of only 0.02% of the native luciferase (Escher, et al., 1989). This is due to the incorrect folding of protein in the presence of the fusion enzyme linker when expressed at high temperature (37 °C). The high temperature of cell culture also causes problem in protein stability. The fusion luciferase often showed a very low expression level in mammalian cells, which was insufficient to develop into a reliable reporter system.
  • Vh fusion luciferase with the two amino acid linker gave a light signal corresponding to only 0.59 fmol of fusion luciferase per mg total protein when it was expressed in mammalian cells (Pazzagli et al, 1992).
  • thermostability bacterial luciferase type should help to increase the stability of enzyme in high culture condition.
  • construction of separated promoters to control the expression of each luxA and luxB gene in eukaryotic cell Koncz et al., 1987; Gupta et al, 2003
  • bi-cystronic expression by placing internal ribosomal entry site between luxA and luxB gene were also carried out (Patterson et al., 2005; Close et al. 2010).
  • LuxA and LuxB proteins were separately translated before assembling to an active enzyme.
  • the inventors according to this invention have engineered a luxA and luxB fusion bacterial luciferase gene and a reporter vector comprising said fusion bacterial luciferase for transfecting into a eukaryotic cell and acting as a suitable eukaryotic or mammalian bioreporter.
  • the bacterial luciferase can be obtained from any luminous bacteria strains that exhibit good thermostability and produce high light intensity.
  • the two luminous bacteria strains, Vibrio campbellii and Photobacterium leiognathi have been used to retrieve the bacterial luciferase by the inventors due to their exhibition of these properties and their availability in Thailand's seashore.
  • the improvement is based on the understanding of bacterial luciferase catalytic mechanism, enzymology and important factors that modulate enzyme activity.
  • the fusion bacterial luciferase reporter system according to this invention provides an alternative means for international scientific community and commercial sectors to utilize the invented fusion bacterial luciferase, the fusion luciferase incorporated reporter vector, and the assay solution as an effective bioluminescence-based assay system for various purposes.
  • the fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention generally comprises a codon optimized luxA and luxB fusion bacterial luciferase gene, a fusion luciferase reporter vector capable of transfecting into a eukaryotic cell or a group of eukaryotic cells wherein the vector comprises a eukaryotic cell-functioning gene cassette sequences and a prokaryotic cell-functioning gene cassette sequences.
  • the luxA and luxB fusion bacterial luciferase gene in the fusion luciferase reporter vector is created by site-directed mutagenesis and is specifically codon optimized for mammalian cell expression.
  • the reporter vector comprising a multiple cloning site (MCS) upstream to the bacterial luciferase gene locale for an insertion of least one promoter or regulation sequence of interest (i.e. as test vector) into said MCS in order to study its effect on gene expression or regulation and/or its response to external compounds, such as therapeutic agents.
  • MCS multiple cloning site
  • a fusion bacterial luciferase assay kit comprising an associating assay solution or assay reagent has also been developed wherein the assay can be used to determine a luciferase expression activity in a cell lysate produced or derived from the cell transfected with the fusion luciferase reporter vector wherein the strength of light emitting signal or bioluminescence emission from the bacterial luciferase activity in the cell lysate after an injection of the assay solution is directly correlated to the strength of the promoter or regulation sequence in controlling a gene expression or in responding to an effect of compound of interest.
  • a general method which has been developed for analyzing gene expression and regulation using the fusion bacterial luciferase comprises the steps of: (1) creating a luxA and luxB fusion bacterial luciferase gene; (2) codon optimizing the fusion bacterial luciferase gene for mammalian cell expression; (3) inserting the codon optimized fusion bacterial luciferase gene into a mammalian cell expression vector to create a fusion bacterial luciferase reporter vector; (4) adding at least one of a promoter or regulation sequence into the reporter vector upstream to the fusion bacterial luciferase gene; (5) transfecting the reporter vector into a cell or a group of cells; (6) obtaining a cell lysate from the cell or group of cells transfected with the reporter vector; (7) creating an assay solution or an assay reagent for adding or injecting to react with the cell lysate; and (8) measuring an expression level as light signal intensity emitted by the bacterial lucifera
  • Figure 1 illustrates the steps of constructing a fusion bacterial luciferase according to this invention by site-directed mutagenesis
  • Figure 2 illustrates the results of thermostability test of the fusion bacterial luciferase from V. campbellii according to this invention at various temperatures (37°C (circles), 40°C (squares) and 45°C (diamonds)).
  • the fusion bacterial luciferase (un labeled symbols) retains thermostability property as the wild-type enzyme (black-labeled symbols).
  • the fusion enzyme displays a half -life at 37°C of -17 hr;
  • Figure 3 illustrates the 3 ⁇ 4 values of fusion and wild-type bacterial luciferases with FMNH- substrate.
  • the 3 ⁇ 4 values of FMNH-binding are at 4.5 ⁇ 0.13 mM and 2.6 ⁇ 0,13 mM for the fusion and wild-type enzymes, respectively.
  • a comparable Kd value indicates that the fusion luciferase retains tight substrate binding property relative to the wild-type enzyme;
  • Figure 4 or SEQ ID NO:l illustrates the nucleotide and amino acid sequences of the codon optimized fusion bacterial luciferase for mammalian cell expression according to this invention;
  • Figure 5 illustrates one embodiment of a promoterless expression vector which can be used as a negative control vector or a test vector.
  • the cassette sequences for functioning in eukayotic cells are on the top row (grey boxes) whereas the cassette sequences for functioning in E. coli are on the bottom part (black boxes);
  • Figure 6 illustrates one embodiment of a positive control vector, namely a S V40 expression vector, wherein a S V40 promoter sequence is inserted (underlined) at the multiple cloning site (MCS) of the promoterless expression vector.
  • the cassette sequences for functioning in eukayotic cells are on the top row (grey boxes) whereas the cassette sequences for functioning in E. coli are on the bottom part (black boxes);
  • Figure 7 illustrates a standard curve of the fusion luciferase according to this invention versus light signal. Activities of various amounts of purified fusion luciferase are measured by luminometer using the optimized assay condition.
  • the standard curve shows linearity over six orders of magnitude from 1.0 amol-10000 frnol of fusion luciferase.
  • the detection limit of the assay was found to be 1.0 amol;
  • Figure 8A illustrates the improvement of cell lysis efficiency using CHAPS as a lysis detergent.
  • HEK293T cells (lxlO 6 ) were resuspended and lysed using different lysis buffers, clarified by centrifugation and measured protein concentrations using Bradford method.
  • Buffer cells were resuspended in 50 mM sodium phosphate buffer pH 7 without lysis process.
  • CL cells were lysed in commercial lysis buffer with one freeze-thaw cycle.
  • 1-FT cells were lysed in 50 mM sodium phosphate buffer pH 7 with one freeze-thaw cycle.
  • 3-FT cells were lysed in 50 mM sodium phosphate buffer pH 7 with three freeze-thaw cycles.0.05% CH: cells were lysed in 50 mM sodium phosphate buffer pH 7 containing 0.05% CHAPS with one freeze-thaw cycle.0.1% CH: cells were lysed in 50 mM sodium phosphate buffer pH 7 containing 0.1% CHAPS with one freeze-thaw cycle. Soni: cells were resuspened in 50 mM sodium phosphate buffer pH 7 and then sonicated;
  • Figure 8B illustrates the effect of detergent on fusion bacterial luciferase (bioluminescence emission) wherein the fusion luciferase activity was determined in the absence and presence of 0.1% v/v detergent; TriX-100; Triton X-100; NP-40; Nonidet P-40; Tw-20; Tween-20; Oct-gly; Octyl glycoside; CHAPS;
  • Figure 9 illustrates the graphs comparing light signals retrieved from the expression of native and codon optimized fusion bacterial luciferase gene in HEK293T and HepG2 cells;
  • Figure 10 illustrates the bioluminescence emission kinetics from bacterial luciferase reaction in the absence and presence of sodium azide;
  • Figure 11 illustrates the graphs comparing signal-to-background ratios of the three luciferase assay systems: bacterial luciferase, firefly luciferase, and renilla luciferase.
  • Fusion bacterial luciferase gene, reporter vector, and assay kit comprises a codon optimized luxA and luxB fusion bacterial luciferase gene, a fusion bacterial luciferase reporter vector capable of transfecting into both the eukaryotic and prokaryotic cells, and a complimentary assay kit of assay reagent.
  • the fusion bacterial luciferase gene according to this invention is created by site-directed mutagenesis and is specifically codon optimized for mammalian cell expression (Figurel).
  • the constructed fusion bacterial luciferase has superior properties of high thermostability and can bind tightly with its substrate, reduced flavin. As illustrated in Figure 2 and Figure 3, the thermostability and binding property to reduced flavin substrate of fusion bacterial luciferase were compared to the wild-type enzyme.
  • the codon optimized gene can enhance the expression efficiency of fusion bacterial luciferase of up to 564 folds in HEK293T cell and 310 folds in HepG2 cell; thus, it is suitable for utilizing as a reporter gene or bioluminescence -based detection system in the eukaryotic cell, preferably mammalian cell.
  • the codon optimized fusion bacterial luciferase gene can be further inserted or incorporated into a mammalian expression vector and transfected into a target cell or a group of cells (i.e. cell culture) wherein the resulting fusion bacterial luciferase reporter vector ( Figure 5) comprises: - a eukaryotic cell-functioning gene cassette sequences (i.e. a set of cassette sequences that are needed for expression of protein in a eukaryotic cell) comprising:
  • -a transcriptional pause sequence for blocking artifact signal from upstream sequence
  • -a multiple cloning site for inserting at least one promoter or regulation sequence of interest
  • -a codon optimized fusion bacterial luciferase gene -a poly A signal sequence for pausing the transcription; and -an enhancer sequence.
  • prokaryotic, preferably Kcoli, cell -functioning gene cassette sequences i.e. a set of cassette sequences that are needed for amplifying the plasmid in E.coli
  • cell -functioning gene cassette sequences i.e. a set of cassette sequences that are needed for amplifying the plasmid in E.coli
  • origin of replication i.e. a set of cassette sequences that are needed for amplifying the plasmid in E.coli
  • antibiotic resistant genes selected from a amplicillin resistant gene or kanamycin resistant gene.
  • a signal sequence can be inserted into the eukaryotic cell-functioning gene portion downstream from the ribosome binding site and linked to the codon optimized fusion bacterial luciferase gene to enable the fusion bacterial luciferase to be continuously secreted into the cell culture medium so that the luciferase activity can be directly measured from the medium sample, not the cell lysate; thus, no cell lysis procedure is further required to break the cells before measurement.
  • the multiple cloning site (MCS) locating on one part of the eukaryotic cell-functioning cassette sequences upstream to the codon optimized fusion bacterial luciferase gene can be used for at least for the purposes of:
  • At least one promoter or regulation sequence of interest i.e. as test vector
  • at least one promoter or regulation sequence of interest i.e. as test vector
  • the fusion bacterial luciferase reporter system further comprises an associating assay solution wherein the assay can be used to determine the luciferase expression activity in the cell lysate produced or derived from the cell transfected with the fusion luciferase reporter vector as described above and wherein one embodiment of the assay solution comprises flavin mononucleotide (FMN), flavin reductase, nicotinamide adenine dinucleotide (NADH) and an aldehyde.
  • FMN flavin mononucleotide
  • NADH nicotinamide adenine dinucleotide
  • the assay solution can be injected into and reacted with the cell lysate containing CI flavin reductase, preferably at the range of 5-500 mU, to determine the luciferase expression activity as a light emitting signal or bioluminescence emission measured and recorded, preferably by a luminometer.
  • reduced FMN one of the bacterial luciferase substrate is generated by the flavin reductase reaction.
  • CI reductase or engineered CI or LuxG FMN oxidoreductase used in this invention has a very high catalytic turnover and high specific activity.
  • bacterial luciferase assay is more efficient with CI reductase and/or engineered CI and/or LuxG FMN oxidoreductase.
  • the assay reagent and condition according to this invention showed a detection limit of 1 amol and displayed a good correlation between light signal and amount of enzyme with a linear response over seven orders of magnitudes from 1 amol- 10000 fmol ( Figure 7).
  • the engineered CI used in this invention was engineered as a truncated form of CI, in which its activity is not controlled by a small molecule called 4-hydroxyphenylacetate (Phongsak et al., 2012).
  • the LuxG FMN oxidoreductase is the flavin reductase enzyme found in the same operon to the bacterial luciferase and physiologically supplies reduced FMN for bacterial luciferase reaction in luminous bacteria (Nijvipakul et al., 2008).
  • the assay solution can be injected into and reacted with the cell lysate containing flavin reductase and fatty acid reductase to determine the luciferase expression activity as a light emitting signal measured and recorded by a luminometer.
  • the second assay composition substitutes aldehyde with aldehyde generation system containing long chain fatty acids (C10-C14), fatty acid reductase, NADPH and ATP.
  • the assay system comprises both the flavin reductase to generate reduced FMN and the fatty acid reductase enzyme in order to convert the long-chain fatty acid to aldehyde to supply the bacterial luciferase reaction.
  • the strength of light emitting signal or bioluminescence from the bacterial luciferase activity in the cell lysate after mixing with the assay solution of either the first or second embodiment as described above is directly correlated to the strength of the promoter or regulation sequence in controlling a gene expression or in responding to an effect of compound of interest.
  • a light signal prolonging agent such as sodium azide or potassium azide can be added into the assay solution to prolong the light signal from 10-15 seconds to at least 50 seconds as demonstrated in Figure 10.
  • the fusion bacterial luciferase reporter system provides a distinctive advantage over firefly luciferase and renilla luciferase systems in that the current fusion bacterial luciferase system exhibits a much lower background noise and, thereby, resulting in a higher signal-to-background ratio as indicated in Figure 11.
  • One experimental result shows that the bacterial luciferase system could generate about 1,800 folds signal-to-background ratio, while with the same expression system, firefly and renilla luciferases displayed only 670 and 19 folds of signal-to-background ratios.
  • the above fusion bacterial luciferase reporter system comprising the codon optimized fusion bacterial luciferase gene, the fusion bacterial luciferase reporter vector, and the bacterial luciferase assay solution and cell lysate according to this invention is generally resulting from a set of experiments carried out by the inventors to construct a luxA and luxB fusion bacterial luciferase gene, to design a mammalian expression vector containing said fusion bacterial luciferase gene (i.e. a fusion bacterial luciferase reporter vector), and to prepare a complimentary or associating bacterial luciferase assay solution and cell lysate.
  • a general method which has been developed by the inventors according to this invention in order to analyze the gene expression and regulation using the fusion bacterial luciferase can be summarized as follows:
  • Bacterial luciferase can be derived from any strains of luminous bacteria available in various parts of the world that exhibit high thermostability and apparent light intensity.
  • the inventors according to this invention preferably to use bacterial luciferase from the two strains: Vibrio campbellii and Photobacterium leiognathi THl.
  • a fusion bacterial luciferase is constructed by using a site-directed mutagenesis technique comprising the following steps: introducing a single nucleotide (G) by primer I immediately upstream of a stop codon of luxA gene to abolish the stop codon, allowing the gene to be read through the intergenic sequence linking the luxA and luxB genes, performing a second mutagenesis by primer ⁇ to transform a second stop codon in the intergenic region between the luxA and luxB genes after adding a single nucleotide from TAA to GAA, and mutating a start codon of the luxB gene from ATG to CAG by primer ⁇ to avoid any internal initiation of translation of luxB gene.
  • the resulting fusion of luxA and luxB enzyme protein consists of ten-amino acid linker including: Valine-Isoleucine-Asparagine-Isoleucine-Phenylalanine-Glutamate-Lysine-Glutamate-Arginine Asparagine (Val-Ile-Asn-Ile-Phe-Glu-Lys-Glu-Arg-Asp).
  • the fusion enzyme exhibits high thermostability as illustrated Figure 2 resulting by incubating the fusion luciferase and the wild- type luciferase in 50mM phosphate buffer at pH 7.0 at various temperature points (37, 40, and 45 oC) and taking the solutions for measurement of their activities at various time points under the spectrofluorometer equipped with a rapid-mixing apparatus whereby the activities of the incubated enzymes at various time points are compared with their starting activities and calculated as percentages of remaining activities. Further, as illustrated in Figure 3, the fusion enzyme also binds tightly to the reduced flavin substrate as the wild-type enzyme by the dissociation constant (Kd) measurement method.
  • Kd dissociation constant
  • the decrease in free reduced FMN (FMNH-) re- oxidation (monitored at 450 nm ⁇ 0.02-10 sec) 20 upon the increased luciferase concentrations is measured to estimate a Kd value for the binding of FMNH- and luciferase.
  • the method generally includes mixing an anaerobic solution FMNH- with air-saturated buffer containing luciferase at various concentrations to re-oxidize free FMNH- and to cause reaction of the luciferase-bound FMN- with molecular oxygen to form a stable C(4a)-peroxyflavin intermediate. As the concentration of luciferase increases, the lesser 25 the free FMNH- oxidation is observed.
  • the decrease in free FMNH- re-oxidation determined from the absorbance change at 450 nm is directly correlated with the FMNH-luciferase complex formation.
  • the Kd value is then calculated from the plot of amplitude changes of absorbance 450 nm versus the free luciferase concentration.
  • Codon optimizing sequence for the fusion bacterial luciferase gene has been obtained from a codon optimizing program which is based on the codon usage frequency of mammalian cell. Codon optimization technique is used to improve the expression efficiency of the fusion bacterial luciferase according to this invention wherein the technique enables nucleotide 5 sequences of fusion luciferase to be replaced with new nucleotide sequences which are more preferential for use in eukaryotic cells, including, but not limited, to mammalian and yeast cells, without changing their protein sequence.
  • the overview of the codon optimized gene sequence of the fusion bacterial luciferase is illustrated in Figure 4 or SEQ ID NO:l.
  • the codon is mammalian cell-optimized for the fusion luciferase gene
  • said gene is then incorporated or inserted into an expression vector wherein the expression vector is designed to have capability to express functional protein in eukaryotic cell and to easily propagate in E. coli.
  • the resulting fusion luciferase gene-incorporated expression vector is also called a fusion luciferase reporter vector.
  • the fusion luciferase reporter vector according to Figure 5 comprises two important parts of the vector: the first part is the cassette sequences that particularly function in eukaryotic cell.
  • This part consists of (1) transcriptional pause sequence for blocking artifact signal from upstream sequence; (2) multiple cloning site (MCS) for inserting interested promoter or regulation sequences; (3) ribosome binding site and signal sequence linked to codon optimized fusion bacterial luciferase (reporter gene); (4) poly A signal sequence to pause the transcription; and (5) enhancer sequence.
  • MCS multiple cloning site
  • the second part of the plasmid is the cassette sequences that particularly function in E. coli, including origin of replication and antibiotic resistant gene.
  • a promoter or regulation sequence of interest can be placed at the upstream site of the fusion luciferase reporter gene.
  • the interested promotor or regulatory fragment can be amplified by PCR and cut with particular restriction enzyme to generate specific ligation sites at both ends.
  • the cut fragment can subsequently be ligated into the multiple cloning site (MCS) or an upstream site of fusion luciferase reporter vector which was previously digested with the same restriction enzyme to PCR fragment.
  • MCS multiple cloning site
  • upstream site of fusion luciferase reporter vector which was previously digested with the same restriction enzyme to PCR fragment.
  • Assessment of the promoter or regulation sequence in driving the gene expression can be carried out by transfecting the constructed vector into mammalian cells, allowing the luciferase gene to be expressed and measuring the luciferase light emission activity, lysing the cells after a period of expression, and measuring the resulting lysate containing bacterial luciferase for luciferase activity by adding the reagents according to the condition provided in the invention.
  • the amount of expressed fusion luciferase measured in terms of light level is directly correlated to the strength of promoter or regulation sequence in controlling the gene expression. Therefore, the strength of promoter or regulation sequence in controlling gene expression or in response to tested conditions can be measured based on the luciferase activity.
  • Figure 6 illustrates a second embodiment of the fusion luciferase reporter vector containing a SV40 promoter sequence (so-called SV40 promoter vector) that has been inserted at the multiple cloning site.
  • the SV40 promoter sequence can be amplified by PCR and can further be ligated into fusion luciferase reporter vector at multiple cloning site (MCS) upstream of the fusion luciferase gene.
  • MCS multiple cloning site
  • the SV40 promoter is well-known to be a strong promoter sequence to enable the fusion luciferase reporter gene to be constitutively expressed inside eukaryotic cells; thus, after transfecting SV40 promoter vector into cell, it is certain that a luciferase activity can be readily observed and so it is suitably used as a positive control.
  • a number of expression tests can be carried out on a target cell or group of cells of interest; these can be on normal, abnormal, cancerous cells, or microbial cells.
  • One example of the expression tests which have been carried out by the inventors is by performing transient transfection wherein the tests begin by plating HEK293T (i.e. human embryonic kidney cell line or HepG2 (i.e.
  • a lysis buffer comprising a phosphate buffer saline pH 7.4 (PBS) and a lysis detergent such as a Zwitterionic detergent, preferably CHAPS or 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate as its effect on promoting fusion bacterial luciferase activity by increasing lysis efficiency ( Figure 8A) and diminishing interference with the light signal emission by fusion bacterial luciferase ( Figure 8B) is clearly shown to be better in comparison to other detergents, pipetting the medium to detach cells, transferring solutions to tubes and centrifuging at cold temperature before washing with ice-cold P
  • the fusion luciferase activity is detected by injecting an assay solution chosen from any of the two compositions as described above.
  • an assay solution chosen from any of the two compositions as described above.
  • the first embodiment comprising FMN, NADH and decanal in sodium phosphate buffer (pH 7.0) can be injected into a cell lysate solution containing CI reductase, and the light signal from the reaction with a luminometer is then recorded.
  • the coupled enzyme, flavin reductase (CI) is stable and has high activity in generating reduced FMN.
  • the assay solution showed a detection limit of 1 amol and displayed a good correlation between light signal and amount of enzyme with a linear response over six orders of magnitude from 1 amol- 10000 fmol of fusion luciferase.
  • the fusion bacterial luciferase reporter system is useful for studying several promoters and regulation sequences including, but not limited to, the following applications:
  • Example 1 For identifying the regulatory element or sequence of the interested gene
  • cyclic AMP is found to up-regulate the expression of pyruvate carboxylase gene and it is required to identify the regulatory element (nucleotide sequence) of the pyruvate carboxylase gene that respond to the cyclic AMP, a suspected regulatory element can be inserted into the upstream of the fusion luciferase gene in the vector and the resulting expression level of fusion luciferase in the presence of cyclic AMP can be detected. If the expression level of the fusion luciferase in the presence of cyclic AMP is higher than no cyclic AMP, the result may suggest that the suspected regulatory sequence that had been inserted upstream of fusion luciferase gene is the regulatory element that respond to cyclic AMP.
  • the suspected regulatory element can be shortened and inserted into the upstream position to compare the expression level of fusion luciferase in the absence and presence of cyclic AMP. If the expression level of fusion luciferase in shorter regulatory element is lower than that of fully regulatory element or no activation of cyclic AMP is present, the result may suggest that the left- out or excluded sequence may be important for cyclic AMP response.
  • Example 2 For identifying company that can have an effect on regulatory element or sequence
  • the known regulatory element of interested gene can be inserted upstream to the fusion luciferase gene in the reporter vector.
  • a test or set of tests is then carried out to identify the effect of specific compound on the expression of fusion luciferase in cell lysate.
  • a compound that can activate or suppress the expression of the gene can be screened via the fusion luciferase reporter system.

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Abstract

A fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention comprising a codon optimized bacterial luciferase gene and a bacterial fusion luciferase reporter vector containing said fusion bacterial luciferase gene wherein the reporter vector is capable of transfecting into a eukaryotic cell or a group of eukaryotic cells. The reporter vector further comprising a multiple cloning site (MCS) upstream to the bacterial luciferase gene locale for an insertion of least one promoter or regulation sequence of interest into said MCS in order to study its effect on gene expression or regulation and/or its response to external compounds, such as therapeutic agents. The invention further comprising an associating assay solution which can be used to determine a luciferase expression activity in a fusion luciferase vector infected cell.

Description

Title of the invention
FUSION BACTERIAL LUCIFERASE GENE, REPORTER VECTOR, AND ASSAY KIT Field of the invention This invention relates to a fusion bacterial luciferase gene, reporter vector, and assay kit wherein the bacterial luciferase gene, specifically a codon optimized luxA and hixB fusion bacterial luciferase, is incorporated into an expression vector and is transfected into a cell or a group of cells of interest. A promoter or regulation sequence of interest can be further incorporated to the vector to investigate its effect on certain gene expression or regulation, or its response to external compound via a bacterial luciferase activity by which a complimentary set of assay kit has been developed to enable the reaction and measurement of said bacterial luciferase expression activity inside the cell sample. Thus, the fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention can be further developed into a high-throughput drug screening system. Background of the invention
Luciferases have been widely used in bioluminescence- based detection technology. From 2010 survey data, nearly 2,000 assays are listed in PubChem database. Thus, about 21% of the available detection methods are relying on bioluminescence- based detection ( Thorne et al. , 2010) . Advantages of bioluminescence- based detection over fluorescence- based systems include no requirement of exogenous excitation light which allows signal generation with low background and eliminates photo-bleaching side effects (Waidman et al., 2011). Thus, luminescence detection typically gives higher sensitivity than fluorescence- based methods. The bioluminescence system is also suitable for measurement of dynamic changes due to its relatively short protein half- life (Allen et al. , 2007) . Therefore, the use of bioluminescence- based bioassays has increased continuously.
Bacterial luciferase produces blue- green light ( max 490 nm) by oxidation of reduced Flavin mononucleotide (FMN) and long chain aldehyde (RCHO) using oxygen (02)
The bioluminescence quantum yield of bacterial luciferase is about 10-16% (Shimomura, 2006; Lei et al., 2004).
FMNH2 + RCHO + 02 +> FMN + RCOOH + H20 + hv (490 nm) Bacterial luciferase is encoded by adjacent duplicated luxA and luxB genes in the lux operon. In luminous bacterial cells, a long chain aldehyde is synthesized by a fatty acid reductase multi- enzymes complex, the gene products of luxC, luxD and luxE in the same lux operon. LuxD is encoded for a transferase catalysing the hydrolysis of fatty acyl group to fatty acid. Synthase and reductase, the gene products of luxC and luxE, consequently catalyse reduction of fatty acid to aldehyde to supply for luciferase reaction. The NADtLFMN oxidoreductase encoded by the luxG gene in the lux operon is responsible for generating majority of reduced FMN to supply the luminescence reaction (Nijvipakul et al., 2008). Bacterial luciferase is a heterodimeric protein and is composed of a- and β-subunits with approximately 77 kDa. These two luciferase subunits share about 30% sequence identity with molecular mass about 40-42 kDa for a-subunit and 36-37 kDa for β-subunit (Szittner et al., 1990).
Several properties allow bacterial luciferase to be a good reporter enzyme in bioluminescence- based detection technology. It is a very sensitive sensor as only about 10 pg of luciferase (Meighen et al., 1991) or 105 luciferase molecules can be detected (Olsson et al., 1989) by a conventional luminometer method. This value is, in several orders of magnitude, lower than other reporter systems such as 3xl09 molecules are required for detection by β-galactosidase, 5-10xl07 molecules for detection by chloramphenicol acytransferase (CAT) and l-3xl08 molecules for detection by alkaline phosphatase (Alam et al., 1990). Luminescence detection is advantageous for the fact that there is usually no endogenous background in non-luminous organisms as compared to the detection by green florescence protein (Waidmann et al., 2011). Luciferase assay is generally fast and requires only a few seconds to process. In addition, light intensity can be correlated well with the amount of luciferase enzyme and mRNA transcripts over a wide range. This is because the luciferase assay reaction undergoes only a single catalytic turnover.
When bacterial luciferase is compared with others bioluminescent reporters, it is considered advantageous due to the low costs of aldehyde and FMN substrates required for luciferase reaction.
The compounds are much cheaper than luciferin and ATP used in firefly luciferase or colelenterazine used in renilla luciferase (Alam et al., 1990). In addition, aldehyde substrate itself can also rapidly traverse the cell membrane (Meighen, 1993). The bacterial luciferase system possesses a distinct advantage over firefly and renilla luciferase systems in that it has a complete set of genes for all substrates synthesis in the same operon, which also allows the system to be self-luminous without cell destruction and exogenous substrate adding if the whole set of genes is expressed.
Bacterial luciferase has been used as a reporter or sensor for detection of various compounds, cell viability and distribution, and in vitro and in vivo metabolic function (Meighen, 1991; Ulitzur,
1997; Greer et al., 2002). However, it was majorly applied in bacterial system. Several attempts have been made to apply bacterial luciferase to be a reporter gene in eukaryotic system but none has shown any promising results because the bacterial luciferase is encoded by two separated luxA and luxB genes which is incompatible to express in eukaryote system. An initial effort to construct the fusion of luxA and luxB genes to enable applications in eukaryotic cells (Olsson et al., 1989;
Boylan, et al., 1989; Werlund-Karlsson et al., 2002) was not successful because the fusion luciferase was extremely sensitive to temperature elevation (Boylan, et al., 1989; Escher, et al.,
1989). With the deca-peptide link, the V. haveyi (Vh) fusion luciferase expressed at 37°C displayed in vivo bioluminescence level in E. coli of only 0.02% of the native luciferase (Escher, et al., 1989). This is due to the incorrect folding of protein in the presence of the fusion enzyme linker when expressed at high temperature (37 °C). The high temperature of cell culture also causes problem in protein stability. The fusion luciferase often showed a very low expression level in mammalian cells, which was insufficient to develop into a reliable reporter system. The Vh fusion luciferase with the two amino acid linker gave a light signal corresponding to only 0.59 fmol of fusion luciferase per mg total protein when it was expressed in mammalian cells (Pazzagli et al, 1992).
Therefore, the using of high thermostability bacterial luciferase type should help to increase the stability of enzyme in high culture condition. To avoid folding problem of fusion enzyme, the construction of separated promoters to control the expression of each luxA and luxB gene in eukaryotic cell (Koncz et al., 1987; Gupta et al, 2003) and bi-cystronic expression by placing internal ribosomal entry site between luxA and luxB gene were also carried out (Patterson et al., 2005; Close et al. 2010). In this type of expression, LuxA and LuxB proteins were separately translated before assembling to an active enzyme. However, this may not be suitable for using as a gene reporter for studying promoter function because the two proteins (LuxA and LuxB) are not controlled by a single promoter. The expression of active luciferase enzyme may not be somewhat directly on promoter strength, and the efficiency of luciferase subunits assembly in eukaryotic cells is not well understood. The mammalian cell codon optimization was also applied to bacterial luciferase to improve its expression efficiency in eukaryotic cell (Patterson et al., 2005; Close et al.2010).
Currently, the inventors according to this invention have engineered a luxA and luxB fusion bacterial luciferase gene and a reporter vector comprising said fusion bacterial luciferase for transfecting into a eukaryotic cell and acting as a suitable eukaryotic or mammalian bioreporter. The bacterial luciferase can be obtained from any luminous bacteria strains that exhibit good thermostability and produce high light intensity. The two luminous bacteria strains, Vibrio campbellii and Photobacterium leiognathi have been used to retrieve the bacterial luciferase by the inventors due to their exhibition of these properties and their availability in Thailand's seashore. The improvement is based on the understanding of bacterial luciferase catalytic mechanism, enzymology and important factors that modulate enzyme activity. The fusion bacterial luciferase reporter system according to this invention provides an alternative means for international scientific community and commercial sectors to utilize the invented fusion bacterial luciferase, the fusion luciferase incorporated reporter vector, and the assay solution as an effective bioluminescence-based assay system for various purposes.
Summary of the invention
The fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention generally comprises a codon optimized luxA and luxB fusion bacterial luciferase gene, a fusion luciferase reporter vector capable of transfecting into a eukaryotic cell or a group of eukaryotic cells wherein the vector comprises a eukaryotic cell-functioning gene cassette sequences and a prokaryotic cell-functioning gene cassette sequences. The luxA and luxB fusion bacterial luciferase gene in the fusion luciferase reporter vector is created by site-directed mutagenesis and is specifically codon optimized for mammalian cell expression. The reporter vector comprising a multiple cloning site (MCS) upstream to the bacterial luciferase gene locale for an insertion of least one promoter or regulation sequence of interest (i.e. as test vector) into said MCS in order to study its effect on gene expression or regulation and/or its response to external compounds, such as therapeutic agents. A fusion bacterial luciferase assay kit comprising an associating assay solution or assay reagent has also been developed wherein the assay can be used to determine a luciferase expression activity in a cell lysate produced or derived from the cell transfected with the fusion luciferase reporter vector wherein the strength of light emitting signal or bioluminescence emission from the bacterial luciferase activity in the cell lysate after an injection of the assay solution is directly correlated to the strength of the promoter or regulation sequence in controlling a gene expression or in responding to an effect of compound of interest.
A general method which has been developed for analyzing gene expression and regulation using the fusion bacterial luciferase comprises the steps of: (1) creating a luxA and luxB fusion bacterial luciferase gene; (2) codon optimizing the fusion bacterial luciferase gene for mammalian cell expression; (3) inserting the codon optimized fusion bacterial luciferase gene into a mammalian cell expression vector to create a fusion bacterial luciferase reporter vector; (4) adding at least one of a promoter or regulation sequence into the reporter vector upstream to the fusion bacterial luciferase gene; (5) transfecting the reporter vector into a cell or a group of cells; (6) obtaining a cell lysate from the cell or group of cells transfected with the reporter vector; (7) creating an assay solution or an assay reagent for adding or injecting to react with the cell lysate; and (8) measuring an expression level as light signal intensity emitted by the bacterial luciferase in the cell lysate mixture after injecting with the assay solution.
Brief description of the drawings Figure 1 illustrates the steps of constructing a fusion bacterial luciferase according to this invention by site-directed mutagenesis;
Figure 2 illustrates the results of thermostability test of the fusion bacterial luciferase from V. campbellii according to this invention at various temperatures (37°C (circles), 40°C (squares) and 45°C (diamonds)). The fusion bacterial luciferase (un labeled symbols) retains thermostability property as the wild-type enzyme (black-labeled symbols). The fusion enzyme displays a half -life at 37°C of -17 hr;
Figure 3 illustrates the ¾ values of fusion and wild-type bacterial luciferases with FMNH- substrate. The ¾ values of FMNH-binding are at 4.5±0.13 mM and 2.6±0,13 mM for the fusion and wild-type enzymes, respectively. A comparable Kd value indicates that the fusion luciferase retains tight substrate binding property relative to the wild-type enzyme; Figure 4 or SEQ ID NO:l illustrates the nucleotide and amino acid sequences of the codon optimized fusion bacterial luciferase for mammalian cell expression according to this invention;
Figure 5 illustrates one embodiment of a promoterless expression vector which can be used as a negative control vector or a test vector. The cassette sequences for functioning in eukayotic cells are on the top row (grey boxes) whereas the cassette sequences for functioning in E. coli are on the bottom part (black boxes);
Figure 6 illustrates one embodiment of a positive control vector, namely a S V40 expression vector, wherein a S V40 promoter sequence is inserted (underlined) at the multiple cloning site (MCS) of the promoterless expression vector. The cassette sequences for functioning in eukayotic cells are on the top row (grey boxes) whereas the cassette sequences for functioning in E. coli are on the bottom part (black boxes);
Figure 7 illustrates a standard curve of the fusion luciferase according to this invention versus light signal. Activities of various amounts of purified fusion luciferase are measured by luminometer using the optimized assay condition. The standard curve shows linearity over six orders of magnitude from 1.0 amol-10000 frnol of fusion luciferase. The detection limit of the assay was found to be 1.0 amol;
Figure 8A illustrates the improvement of cell lysis efficiency using CHAPS as a lysis detergent. HEK293T cells (lxlO6) were resuspended and lysed using different lysis buffers, clarified by centrifugation and measured protein concentrations using Bradford method. Buffer: cells were resuspended in 50 mM sodium phosphate buffer pH 7 without lysis process. CL: cells were lysed in commercial lysis buffer with one freeze-thaw cycle. 1-FT: cells were lysed in 50 mM sodium phosphate buffer pH 7 with one freeze-thaw cycle. 3-FT: cells were lysed in 50 mM sodium phosphate buffer pH 7 with three freeze-thaw cycles.0.05% CH: cells were lysed in 50 mM sodium phosphate buffer pH 7 containing 0.05% CHAPS with one freeze-thaw cycle.0.1% CH: cells were lysed in 50 mM sodium phosphate buffer pH 7 containing 0.1% CHAPS with one freeze-thaw cycle. Soni: cells were resuspened in 50 mM sodium phosphate buffer pH 7 and then sonicated;
Figure 8B illustrates the effect of detergent on fusion bacterial luciferase (bioluminescence emission) wherein the fusion luciferase activity was determined in the absence and presence of 0.1% v/v detergent; TriX-100; Triton X-100; NP-40; Nonidet P-40; Tw-20; Tween-20; Oct-gly; Octyl glycoside; CHAPS;
Figure 9 illustrates the graphs comparing light signals retrieved from the expression of native and codon optimized fusion bacterial luciferase gene in HEK293T and HepG2 cells; Figure 10 illustrates the bioluminescence emission kinetics from bacterial luciferase reaction in the absence and presence of sodium azide;
Figure 11 illustrates the graphs comparing signal-to-background ratios of the three luciferase assay systems: bacterial luciferase, firefly luciferase, and renilla luciferase.
Detailed description of the invention Fusion bacterial luciferase gene, reporter vector, and assay kit according to this invention comprises a codon optimized luxA and luxB fusion bacterial luciferase gene, a fusion bacterial luciferase reporter vector capable of transfecting into both the eukaryotic and prokaryotic cells, and a complimentary assay kit of assay reagent.
The fusion bacterial luciferase gene according to this invention is created by site-directed mutagenesis and is specifically codon optimized for mammalian cell expression (Figurel). The constructed fusion bacterial luciferase has superior properties of high thermostability and can bind tightly with its substrate, reduced flavin. As illustrated in Figure 2 and Figure 3, the thermostability and binding property to reduced flavin substrate of fusion bacterial luciferase were compared to the wild-type enzyme. The results suggest the comparability of thermostability and binding property of the fusion luciferase to those of the wild-type which has been known to have much superior properties in flavin binding and thermostability as compared to the \.harveyi enzyme (Suadee et al., 2007). The gene sequencing of said codon optimized gene is as set forth in Figure 4 or SEQ ID NO:l. As illustrated in Figure 9, the codon optimized gene can enhance the expression efficiency of fusion bacterial luciferase of up to 564 folds in HEK293T cell and 310 folds in HepG2 cell; thus, it is suitable for utilizing as a reporter gene or bioluminescence -based detection system in the eukaryotic cell, preferably mammalian cell.
The codon optimized fusion bacterial luciferase gene can be further inserted or incorporated into a mammalian expression vector and transfected into a target cell or a group of cells (i.e. cell culture) wherein the resulting fusion bacterial luciferase reporter vector (Figure 5) comprises: - a eukaryotic cell-functioning gene cassette sequences (i.e. a set of cassette sequences that are needed for expression of protein in a eukaryotic cell) comprising:
-a transcriptional pause sequence for blocking artifact signal from upstream sequence; -a multiple cloning site (MCS) for inserting at least one promoter or regulation sequence of interest;
-a ribosome binding site;
-a codon optimized fusion bacterial luciferase gene; -a poly A signal sequence for pausing the transcription; and -an enhancer sequence.
- a prokaryotic, preferably Kcoli, cell -functioning gene cassette sequences (i.e. a set of cassette sequences that are needed for amplifying the plasmid in E.coli) comprising origin of replication and antibiotic resistant genes selected from a amplicillin resistant gene or kanamycin resistant gene. A signal sequence can be inserted into the eukaryotic cell-functioning gene portion downstream from the ribosome binding site and linked to the codon optimized fusion bacterial luciferase gene to enable the fusion bacterial luciferase to be continuously secreted into the cell culture medium so that the luciferase activity can be directly measured from the medium sample, not the cell lysate; thus, no cell lysis procedure is further required to break the cells before measurement. The multiple cloning site (MCS) locating on one part of the eukaryotic cell-functioning cassette sequences upstream to the codon optimized fusion bacterial luciferase gene can be used for at least for the purposes of:
-inserting at least one promoter or regulation sequence of interest (i.e. as test vector) to study its effect on gene expression or regulation and/or its response to external compounds, such as therapeutic agents;
-inserting a SV40 promoter to enable the vector to act as a positive control vector (as shown in Figure 6) so that if the SV40 vector is transfected in a cell, the luciferase activity measured by emitted light is ensured in a cell lysate which derived from the transfected cell or group of cells; or -leaving the MCS upstream of the fusion bacterial luciferase gene unoccupied to enable the vector to act as a negative control wherein no luciferase activity shall be expected in cell lysate as illustrated in Figure 5.
The fusion bacterial luciferase reporter system according to this invention further comprises an associating assay solution wherein the assay can be used to determine the luciferase expression activity in the cell lysate produced or derived from the cell transfected with the fusion luciferase reporter vector as described above and wherein one embodiment of the assay solution comprises flavin mononucleotide (FMN), flavin reductase, nicotinamide adenine dinucleotide (NADH) and an aldehyde. In this embodiment, the assay solution can be injected into and reacted with the cell lysate containing CI flavin reductase, preferably at the range of 5-500 mU, to determine the luciferase expression activity as a light emitting signal or bioluminescence emission measured and recorded, preferably by a luminometer. In this coupling assay reaction, reduced FMN, one of the bacterial luciferase substrate is generated by the flavin reductase reaction. Preferably CI reductase or engineered CI or LuxG FMN oxidoreductase used in this invention has a very high catalytic turnover and high specific activity. Therefore, bacterial luciferase assay is more efficient with CI reductase and/or engineered CI and/or LuxG FMN oxidoreductase. The assay reagent and condition according to this invention, showed a detection limit of 1 amol and displayed a good correlation between light signal and amount of enzyme with a linear response over seven orders of magnitudes from 1 amol- 10000 fmol (Figure 7). The engineered CI used in this invention was engineered as a truncated form of CI, in which its activity is not controlled by a small molecule called 4-hydroxyphenylacetate (Phongsak et al., 2012). The LuxG FMN oxidoreductase is the flavin reductase enzyme found in the same operon to the bacterial luciferase and physiologically supplies reduced FMN for bacterial luciferase reaction in luminous bacteria (Nijvipakul et al., 2008). The second embodiment of the assay solution which can be used to determine a luciferase expression activity in a cell lysate produced or derived from the cell transfected with the fusion luciferase reporter vector comprises of flavin reductase, FMN, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), fatty acid reductase, long chain fatty acid (C10-C14), adenosine triphosphate (ATP). In such embodiment, the assay solution can be injected into and reacted with the cell lysate containing flavin reductase and fatty acid reductase to determine the luciferase expression activity as a light emitting signal measured and recorded by a luminometer. Due to the inherently unstable and toxicity of aldehyde substrate as represented by decanal (CIO) or dodecanal (C12) or tetradecanal (C14), the second assay composition substitutes aldehyde with aldehyde generation system containing long chain fatty acids (C10-C14), fatty acid reductase, NADPH and ATP. The assay system comprises both the flavin reductase to generate reduced FMN and the fatty acid reductase enzyme in order to convert the long-chain fatty acid to aldehyde to supply the bacterial luciferase reaction. The strength of light emitting signal or bioluminescence from the bacterial luciferase activity in the cell lysate after mixing with the assay solution of either the first or second embodiment as described above is directly correlated to the strength of the promoter or regulation sequence in controlling a gene expression or in responding to an effect of compound of interest. Optionally, to prolong the light emitting signal of said bacterial luciferase reaction, a light signal prolonging agent, such as sodium azide or potassium azide can be added into the assay solution to prolong the light signal from 10-15 seconds to at least 50 seconds as demonstrated in Figure 10.
The fusion bacterial luciferase reporter system according to this invention provides a distinctive advantage over firefly luciferase and renilla luciferase systems in that the current fusion bacterial luciferase system exhibits a much lower background noise and, thereby, resulting in a higher signal-to-background ratio as indicated in Figure 11. One experimental result shows that the bacterial luciferase system could generate about 1,800 folds signal-to-background ratio, while with the same expression system, firefly and renilla luciferases displayed only 670 and 19 folds of signal-to-background ratios.
The above fusion bacterial luciferase reporter system comprising the codon optimized fusion bacterial luciferase gene, the fusion bacterial luciferase reporter vector, and the bacterial luciferase assay solution and cell lysate according to this invention is generally resulting from a set of experiments carried out by the inventors to construct a luxA and luxB fusion bacterial luciferase gene, to design a mammalian expression vector containing said fusion bacterial luciferase gene (i.e. a fusion bacterial luciferase reporter vector), and to prepare a complimentary or associating bacterial luciferase assay solution and cell lysate. A general method which has been developed by the inventors according to this invention in order to analyze the gene expression and regulation using the fusion bacterial luciferase can be summarized as follows:
- creating a luxA and luxB fusion bacterial luciferase gene;
- codon optimizing the fusion bacterial luciferase gene for mammalian cell expression; -inserting the codon optimized fusion bacterial luciferase gene into a mammalian cell expression vector to create a fusion bacterial luciferase reporter vector; - adding at least one of a promoter or regulation sequence into the reporter vector upstream to the fusion bacterial luciferase gene;
- transfecting the reporter vector into a cell or a group of cells in order to enable the promoter or regulation sequence-driven fusion bacterial luciferase gene to be express; - obtaining a cell lysate from the cell or group of cells transfected with the reporter vector;
- creating an assay solution or an assay reagent for adding or injecting into and reacting with the cell lysate; and
- measuring an expression level as light signal intensity emitted by the bacterial luciferase in the cell mixture after injecting with the assay solution. Further details with regards to the method of analyzing gene expression and regulation using the fusion bacterial luciferase as described above are best accompanied by the experimental data set and their results as follows:
1. Contracting a luxA and luxB fusion bacterial luciferase gene:
Bacterial luciferase can be derived from any strains of luminous bacteria available in various parts of the world that exhibit high thermostability and apparent light intensity. The inventors according to this invention preferably to use bacterial luciferase from the two strains: Vibrio campbellii and Photobacterium leiognathi THl. As illustrated in Figure 1, a fusion bacterial luciferase according to this invention is constructed by using a site-directed mutagenesis technique comprising the following steps: introducing a single nucleotide (G) by primer I immediately upstream of a stop codon of luxA gene to abolish the stop codon, allowing the gene to be read through the intergenic sequence linking the luxA and luxB genes, performing a second mutagenesis by primer Π to transform a second stop codon in the intergenic region between the luxA and luxB genes after adding a single nucleotide from TAA to GAA, and mutating a start codon of the luxB gene from ATG to CAG by primer ΠΙ to avoid any internal initiation of translation of luxB gene. The resulting fusion of luxA and luxB enzyme protein consists of ten-amino acid linker including: Valine-Isoleucine-Asparagine-Isoleucine-Phenylalanine-Glutamate-Lysine-Glutamate-Arginine Asparagine (Val-Ile-Asn-Ile-Phe-Glu-Lys-Glu-Arg-Asp). The fusion enzyme exhibits high thermostability as illustrated Figure 2 resulting by incubating the fusion luciferase and the wild- type luciferase in 50mM phosphate buffer at pH 7.0 at various temperature points (37, 40, and 45 oC) and taking the solutions for measurement of their activities at various time points under the spectrofluorometer equipped with a rapid-mixing apparatus whereby the activities of the incubated enzymes at various time points are compared with their starting activities and calculated as percentages of remaining activities. Further, as illustrated in Figure 3, the fusion enzyme also binds tightly to the reduced flavin substrate as the wild-type enzyme by the dissociation constant (Kd) measurement method. In this method, the decrease in free reduced FMN (FMNH-) re- oxidation (monitored at 450 nm ~0.02-10 sec) 20 upon the increased luciferase concentrations is measured to estimate a Kd value for the binding of FMNH- and luciferase. The method generally includes mixing an anaerobic solution FMNH- with air-saturated buffer containing luciferase at various concentrations to re-oxidize free FMNH- and to cause reaction of the luciferase-bound FMN- with molecular oxygen to form a stable C(4a)-peroxyflavin intermediate. As the concentration of luciferase increases, the lesser 25 the free FMNH- oxidation is observed. Thus, the decrease in free FMNH- re-oxidation determined from the absorbance change at 450 nm is directly correlated with the FMNH-luciferase complex formation. The Kd value is then calculated from the plot of amplitude changes of absorbance 450 nm versus the free luciferase concentration.
2. Creating a codon optimized fusion bacterial luciferase gene: Codon optimizing sequence for the fusion bacterial luciferase gene has been obtained from a codon optimizing program which is based on the codon usage frequency of mammalian cell. Codon optimization technique is used to improve the expression efficiency of the fusion bacterial luciferase according to this invention wherein the technique enables nucleotide 5 sequences of fusion luciferase to be replaced with new nucleotide sequences which are more preferential for use in eukaryotic cells, including, but not limited, to mammalian and yeast cells, without changing their protein sequence. The overview of the codon optimized gene sequence of the fusion bacterial luciferase is illustrated in Figure 4 or SEQ ID NO:l.
3. Designing an expression vector containing fusion bacterial luciferase gene: 10
After the codon is mammalian cell-optimized for the fusion luciferase gene, said gene is then incorporated or inserted into an expression vector wherein the expression vector is designed to have capability to express functional protein in eukaryotic cell and to easily propagate in E. coli. The resulting fusion luciferase gene-incorporated expression vector is also called a fusion luciferase reporter vector. In one embodiment of the fusion luciferase reporter vector according to Figure 5 comprises two important parts of the vector: the first part is the cassette sequences that particularly function in eukaryotic cell. This part consists of (1) transcriptional pause sequence for blocking artifact signal from upstream sequence; (2) multiple cloning site (MCS) for inserting interested promoter or regulation sequences; (3) ribosome binding site and signal sequence linked to codon optimized fusion bacterial luciferase (reporter gene); (4) poly A signal sequence to pause the transcription; and (5) enhancer sequence. The second part of the plasmid is the cassette sequences that particularly function in E. coli, including origin of replication and antibiotic resistant gene. For the study of gene expression and regulation, a promoter or regulation sequence of interest can be placed at the upstream site of the fusion luciferase reporter gene. The interested promotor or regulatory fragment can be amplified by PCR and cut with particular restriction enzyme to generate specific ligation sites at both ends. The cut fragment can subsequently be ligated into the multiple cloning site (MCS) or an upstream site of fusion luciferase reporter vector which was previously digested with the same restriction enzyme to PCR fragment. Assessment of the promoter or regulation sequence in driving the gene expression can be carried out by transfecting the constructed vector into mammalian cells, allowing the luciferase gene to be expressed and measuring the luciferase light emission activity, lysing the cells after a period of expression, and measuring the resulting lysate containing bacterial luciferase for luciferase activity by adding the reagents according to the condition provided in the invention. The amount of expressed fusion luciferase measured in terms of light level is directly correlated to the strength of promoter or regulation sequence in controlling the gene expression. Therefore, the strength of promoter or regulation sequence in controlling gene expression or in response to tested conditions can be measured based on the luciferase activity. Figure 6 illustrates a second embodiment of the fusion luciferase reporter vector containing a SV40 promoter sequence (so-called SV40 promoter vector) that has been inserted at the multiple cloning site. The SV40 promoter sequence can be amplified by PCR and can further be ligated into fusion luciferase reporter vector at multiple cloning site (MCS) upstream of the fusion luciferase gene. In this embodiment, the SV40 promoter is well-known to be a strong promoter sequence to enable the fusion luciferase reporter gene to be constitutively expressed inside eukaryotic cells; thus, after transfecting SV40 promoter vector into cell, it is certain that a luciferase activity can be readily observed and so it is suitably used as a positive control.
4. Transfecting a cell or group of cells with the fusion luciferase reporter vector: 20
After the fusion luciferase reporter vector has been created, a number of expression tests can be carried out on a target cell or group of cells of interest; these can be on normal, abnormal, cancerous cells, or microbial cells. One example of the expression tests which have been carried out by the inventors is by performing transient transfection wherein the tests begin by plating HEK293T (i.e. human embryonic kidney cell line or HepG2 (i.e. human 25 hepatocyte carcinoma cell line) cells and culturing in antibiotic-free medium in well plates, transfecting the cells by mixing plasmid with a mediator or transfecting agent such as lipofectamine TM2000 in Opti- MEM I reduced serum medium, maintaining the transfected cells for 24, 48 and 72 h before harvesting them by adding a lysis buffer comprising a phosphate buffer saline pH 7.4 (PBS) and a lysis detergent such as a Zwitterionic detergent, preferably CHAPS or 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate as its effect on promoting fusion bacterial luciferase activity by increasing lysis efficiency (Figure 8A) and diminishing interference with the light signal emission by fusion bacterial luciferase (Figure 8B) is clearly shown to be better in comparison to other detergents, pipetting the medium to detach cells, transferring solutions to tubes and centrifuging at cold temperature before washing with ice-cold PBS, re-suspending cell pellet in sodium phosphate buffer pH 7.0 and subjecting to cycles of freeze-thaw process, and obtaining cell lysate by centrifugation whereby the resulting total protein concentration is then determined by Bradford's assay, and the cell lysate can further being used to assay for luciferase activity.
5. Assaying for luciferase activity:
Following the expression tests above, the fusion luciferase activity is detected by injecting an assay solution chosen from any of the two compositions as described above. For example, the first embodiment comprising FMN, NADH and decanal in sodium phosphate buffer (pH 7.0) can be injected into a cell lysate solution containing CI reductase, and the light signal from the reaction with a luminometer is then recorded. The coupled enzyme, flavin reductase (CI), is stable and has high activity in generating reduced FMN. According to the standard curve of fusion luciferase versus light signal according to Figure 7, the assay solution showed a detection limit of 1 amol and displayed a good correlation between light signal and amount of enzyme with a linear response over six orders of magnitude from 1 amol- 10000 fmol of fusion luciferase.
The fusion bacterial luciferase reporter system is useful for studying several promoters and regulation sequences including, but not limited to, the following applications:
Example 1: For identifying the regulatory element or sequence of the interested gene
If cyclic AMP is found to up-regulate the expression of pyruvate carboxylase gene and it is required to identify the regulatory element (nucleotide sequence) of the pyruvate carboxylase gene that respond to the cyclic AMP, a suspected regulatory element can be inserted into the upstream of the fusion luciferase gene in the vector and the resulting expression level of fusion luciferase in the presence of cyclic AMP can be detected. If the expression level of the fusion luciferase in the presence of cyclic AMP is higher than no cyclic AMP, the result may suggest that the suspected regulatory sequence that had been inserted upstream of fusion luciferase gene is the regulatory element that respond to cyclic AMP. Further, the suspected regulatory element can be shortened and inserted into the upstream position to compare the expression level of fusion luciferase in the absence and presence of cyclic AMP. If the expression level of fusion luciferase in shorter regulatory element is lower than that of fully regulatory element or no activation of cyclic AMP is present, the result may suggest that the left- out or excluded sequence may be important for cyclic AMP response. Example 2: For identifying company that can have an effect on regulatory element or sequence
To study or to screen the effect of certain compound (such as a therapeutic compound) on the known regulatory element of interested gene, the known regulatory element of interested gene can be inserted upstream to the fusion luciferase gene in the reporter vector. A test or set of tests is then carried out to identify the effect of specific compound on the expression of fusion luciferase in cell lysate. With this method, a compound that can activate or suppress the expression of the gene can be screened via the fusion luciferase reporter system.

Claims

Claims
1. A fusion bacterial luciferase gene comprising a luxA and luxB fusion bacterial luciferase gene wherein the gene is codon optimized for a mammalian cell expression and its gene sequencing is as set forth in SEQ ID NO: 1.
2. A fusion bacterial luciferase reporter vector capable of transfecting into a cell or a group of cells, wherein the vector comprises:
a eukaryotic cell-functioning gene cassette sequences comprising:
- a transcriptional pause sequence for blocking artifact signal from upstream sequence;
- a multiple cloning site (MCS) for inserting at least one promoter or regulation sequence of interest;
- a ribosome binding site;
- a codon optimized luxA and luxB fusion bacterial luciferase gene;
- a poly A signal sequence for pausing the transcription; and
- an enhancer sequence.
a prokaryotic cell-functioning gene cassette sequences, comprising origin of replication and antibiotic resistant genes selected from a amplicillin resistant gene or kanamycin resistant gene.
3. The fusion bacterial luciferase reporter vector according to claim 2, wherein a signal sequence locating downstream from the ribosome binding site in the eukaryotic cell-functioning gene cassette sequences is linking to the codon optimized luxA and luxB fusion bacterial luciferase gene.
4. The fusion bacterial luciferase reporter vector according to claim 2 wherein the prokaryotic cell- functioning cassette sequences are effective in E.coli.
5. The fusion bacterial luciferase reporter vector according to claim 2 wherein the luxA and luxB fusion bacterial luciferase gene is codon optimized for a mammalian cell expression and the gene sequencing of said codon optimized gene is as set forth in SEQ ID NO:l.
6. The fusion bacterial luciferase reporter vector according to claim 2 wherein at least one promoter or regulation sequence is inserted into the MCS upstream to the luxA and luxB fusion bacterial luciferase gene.
7. The fusion bacterial luciferase reporter vector according to claim 2 wherein a SV40 promoter is inserted into the MCS upstream to the luxA and luxB fusion bacterial luciferase gene for the fusion luciferase reporter vector to act as a positive control vector.
8. The fusion bacterial lucif erase reporter vector according to claim 2 wherein the MCS upstream to the luxA and luxB fusion bacterial luciferase gene is without a promoter or regulation sequence for the fusion luciferase reporter vector to act as a negative control vector.
9. The fusion bacterial luciferase reporter vector according to claim 2 wherein the cell is chosen from a eukaryotic cell or a prokaryotic cell.
10. The fusion bacterial luciferase reporter vector according to claim 2 wherein the cell is a mammalian cell.
11. A fusion bacterial luciferase assay solution capable of reacting with a cell lysate deriving from a cell or a group of cells transfected with a fusion luciferase reporter vector wherein said assay solution comprises a flavin reductase, a flavin mononucleotide (FMN), a nicotinamide adenine dinucleotide (NADH), and an aldehyde.
12. A fusion bacterial luciferase assay solution capable of reacting with a cell lysate deriving from a cell or a group of cells transfected with a fusion luciferase reporter vector wherein said assay solution comprises 50-500 mU of a flavin reductase, 2.5-50 μΜ of flavin mononucleotide (FMN), 50-200 μΜ of nicotinamide adenine dinucleotide (NADH), and 5-20 μΜ of aldehyde.
13. The fusion bacterial luciferase assay solution according to claim 11 or 12 wherein the aldehyde is selected from one of decanal, dodecanal and tetradecanal.
14. A fusion bacterial luciferase assay solution capable of reacting with a cell lysate deriving from a cell or a group of cells transfected with a fusion luciferase reporter vector wherein said assay solution comprises a flavin reductase, a flavin mononucleotide (FMN), a nicotinamide adenine dinucleotide (NADH), a fatty acid reductase, a nicotinamide adenine dinucleotide phosphate (NADPH), a long chain fatty acid, adenosine triphosphate (ATP).
15. A fusion bacterial luciferase assay solution capable of reacting with a cell lysate deriving from a cell or a group of cells transfected with a fusion luciferase reporter vector wherein said assay solution comprises 50-500 mU of a flavin reductase, 2.5-50 μΜ of a flavin mononucleotide (FMN), 50-200 μΜ of nicotinamide adenine dinucleotide (NADH), 0.1-100 μg of a fatty acid reductase, 50 μΜ-10 mM of a nicotinamide adenine dinucleotide phosphate (NADPH), 1-50 μΜ of a long chain fatty acid, and 50 μΜ-10 mM of an adenosine triphosphate (ATP).
16. The fusion bacterial luciferase assay solution according to any of claims 14 or 15 wherein the long chain fatty acid is selected from one of decanoic acid, dodecanoic acid and tetradecanoic acid.
17. The fusion bacterial luciferase assay solution according to any of claim 11,12,14 or 15 wherein the flavin reductase is selected from one of CI reductase, an engineered CI reductase and a LuxG FMN oxidoresuctase.
18. The fusion bacterial luciferase assay solution according to any of claims 11, 12, 14 or 15 wherein the fusion luciferase reporter vector comprises:
a eukaryotic cell-functioning gene cassette sequences comprising:
- a transcriptional pause sequence for blocking artifact signal from upstream sequence; - a multiple cloning site (MCS) for inserting at least one promoter or regulation sequence of interest;
- a ribosome binding site;
- a codon optimized luxA and luxB fusion bacterial luciferase gene;
- a poly A signal sequence for pausing the transcription; and
- an enhancer sequence.
a prokaryotic cell-functioning gene cassette sequences, comprising origin of replication and antibiotic resistant genes selected from a amplicillin resistant gene or kanamycin resistant gene.
19. The fusion bacterial luciferase assay solution according to claims 18 wherein a signal sequence locating downstream from the ribosome binding site in the eukaryotic cell-functioning gene cassette sequences is linking to the codon optimized luxA and luxB fusion
bacterial luciferase gene is as set forth in SEQ ID NO:l
20. The fusion bacterial luciferase assay solution according to claims 18 wherein the codon optimized luxA and luxB fusion bacterial luciferase gene is use for mammalian cell expression and the gene sequencing of the codon optimized luxA and luxB fusion bacterial luciferase gene is as set forth in SEQ ID NO: 1
21. The fusion bacterial luciferase assay solution according to claim 11, 12, 14 or 15 further comprising a light signal prolonging agent chosen from a sodium azide or a potassium azide.
22. The fusion bacterial luciferase assay solution according to any of claims 11, 12, 14 or 15 wherein the cell lysate is collected from the lysing of the transfected cells by freeze- thaw in a buffer containing a zwitterionic detergent.
23. The fusion bacterial luciferase assay solution according to claim 22 wherein the zwitterionic detergent is CHAPS or 3- [ ( 3- cholamidopropyl) dimethylammonio] - 1- propanesulfonate.
24. The fusion bacterial luciferase assay solution according to claim 23 wherein the CHAPS has a concentration of between 0.05-0.5% w v.
PCT/TH2016/000051 2016-06-03 2016-06-03 Fusion bacterial luciferase gene, reporter vector, and assay kit Ceased WO2017209700A1 (en)

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