Patent Application
20170158742
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 6,111 Byte ASCII (Text) file named “727306_ST25,” dated Dec. 13, 2016.
The present invention relates to a biotinylated protein. In particular, it relates to biotinylated flavivirus structural and non-structural proteins.
The family Flaviviridae contains at least 70 arthropod-transmitted viruses, many of which infect humans and other vertebrates. Dengue virus (DENV) belongs to the Flavivirus genus within the Flaviviridae family. Other members of the Flavivirus genus include Yellow Fever virus (YFV), West Nile virus (WNV), Kunjin virus (KUNV), Japanese Encephalitis virus (JEV), and Tick-Borne Encephalitis virus (TBEV), just to name a few. All flaviviruses, including West Nile Virus, St Louis encephalitis, dengue, Japanese encephalitis, yellow fever and Kunjin viruses share similar size, symmetry and appearance. Despite the fact that flaviviruses may use different process to enter a host cell, such as endocytosis (described for West Nile Virus and Kunjin Virus) and direct fusion of the cell (described for dengue and Encephalitis Virus), entry of all flaviviruses into the host-cell involves an interaction between the virus and a receptor of the cell.
DENV infection encompasses a wide spectrum of severity ranging from mild asymptomatic Dengue Fever (DF) to critical and fatal Dengue Haemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS). DENV causes about 15,000 deaths annually and it is estimated that more than 2.5 billion people are at risk of DENV infection in more than 100 countries (Gubler, 2002). However, anti-viral drug and vaccine are yet to be available in the market.
The whole mature virion particle is about 50 nm in diameter. Each virion contains a single positive-stranded RNA that is encapsulated by multiple copies of capsid (C) protein to form a spherical cage-like structure called nucleocapsid or core. The core is approximately 30 nm in diameter which can be seen as a dense particle under electron microscope (Hase et al., 1987). Nucleocapsid is surrounded by a 10 nm thick lipid bilayer derived from host cell membrane anchored with 180 membrane (M) protein and 180 envelope (E) protein (Zhang et al., 2003).
DENV C protein is the first structural protein found in the open reading frame (ORF) of its genome. It is one of the three structural proteins that form a mature virus. Although the sequence homology of C proteins among other flaviviruses is poorly conserved (
Other than its structural function, DENV C protein is also known to carry non-structural functions. It is currently believed that multifunctional C protein is essential in the viral replication, assembly, RNA encapsidation, as well as pathogenesis. The role of C protein in inducing apoptosis by activating caspase-3 and caspase-9 leading to mitochondrial dysfunction was reported (Yang et al., 2008). Recently, we discovered that WNV and DENV C protein interact with human Sec3 exocyst protein (hSec3p) to antagonize the antiviral effect caused by hSec3p (Bhuvanakantham et al., 2010). Moreover, DENV C protein was also known to localize in the nucleus of infected cells during replication (Tadano et al., 1989). We found that C protein was transported into the nucleus by importain-α/β complex and the loss of the nuclear translocation ability affected virus replication (Bhuvanakantham et al., 2009). However, the exact role of C protein in the nucleus still remains unclear and further investigation is warrant.
There is also a lack of information about the interaction of DENV C protein with either prM and E proteins or even its RNA genome. The binding of NS2B-NS3 protein with C protein and how it cleaves C-prM polyprotein is also unclear. This is mainly due to the difficulty of expressing and purifying full-length C protein for functional and structural studies. The NMR structure of DENV C protein studied thus far only encompasses 80 residues from 21st to 100th amino acid residues (Ma et al., 2004). The N-terminus is removed from the structural study because it is conformationally labile and unstable (Jones et al., 2003). The hydrophobic C-terminus of DENV C protein is also excluded due to its solubility problem. Nevertheless, the N-terminus of C protein is found to be antigenic as predicted bioinformatically (
Biotinylation is a popular process in protein engineering to ease detection and purification (Chapman-Smith and Cronan, 1999, Cull and Schatz, 2000). Biotinylated hemagglutinin, for instance, was generated to develop a subtype-specific serological assay to diagnose influenza A virus in patients' sera (Postel, Letzel, Muller, Ehricht, Pourquier, Dauber, Grund, Beer and Harder, 2011). One of the advantages of using biotinylated protein is that the detection sensitivity of the biotinylated protein is greatly enhanced by the high affinity and specificity between biotin and streptavidin (Bayer and Wilchek, 1990). This advantage is exploited for techniques such as co-immunoprecipitation or library screening of interaction proteome (He et al., 2009, Markham et al., 2007, Moreland et al., 2010). Not only that, site-specific biotinylation was also developed as a means for molecular labeling and imaging (Howarth and Ting, 2008, Sueda et al., 2011).
There are various approaches to biotinylate a protein, either chemically or enzymatically (Cull and Schatz, 2000). Various commercial kits are available in the market for chemical biotinylation by conjugating biotin molecules on proteins or antibodies that contain primary amines. However, chemical biotinylation may lead to non-specific and non-homogenous incorporation of biotin which might result in possible loss of activity of the protein (Bayer and Wilchek, 1990). Moreover, another step of removing the excess biotin reagent will increase the chance of losing more proteins.
The challenge of studying this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively-charged, stability and solubility properties. Full-length DENV C protein was expressed and purified for structural and functional studies. Since biotinylation has been widely used to date for purification, detection, diagnostic, protein-protein interaction studies, imaging studies, and other molecular studies (Chapman-Smith and Cronan, 1999, de Boer et al., 2003, Howarth and Ting, 2008, Postel et al., 2011, Qi and Katagiri, 2011), a biotinylation site was engineered into C protein to ease further downstream experiments. Using expression and purification strategies, the present inventors have successfully obtained functional biotinylated full-length DENV C protein.
In particular, the present inventors have developed a strategy to construct, express, biotinylate and purify non-truncated, full-length DENV C protein. A 6×His tag and a biotin acceptor peptide (BAP) were cloned at the N-terminus of C protein using overlapping extension-PCR (OE-PCR) method for site-specific biotinylation. The final construct was inserted into pET28a plasmid and BL-21 (CodonPlus) expression competent cell strain was selected as there are 12% rare codons in the C protein sequence. Strikingly, and advantageously, the inventors found that the recombinant proteins with BAP were biotinylated endogenously with high efficiency in Escherichia coli BL-21 strains. To purify this his-tagged C protein, nickel-nitriloacetic acid (Ni-NTA) affinity chromatography was first carried out under denaturing condition. After stepwise dialysis and concurrent refolding, ion exchange-fast protein liquid chromatography (IEX-FPLC) was performed to further separate the residual contaminants. To obtain C protein with high purity, final round of purification with size exclusion chromatography was carried out and a single peak corresponding to C protein was attained. With this optimized sequential purification protocol, the inventors successfully generated pure biotinylated full-length DENV C protein. The functionality of this purified non-truncated DENV C protein was examined and it was found to be suitable for structural and molecular studies.
Advantageously, biotinylated DENV C protein is useful for various cellular, molecular and imaging experiments because of the high detection limit while the function of the protein is still well preserved.
Still advantageously, the fused protein may be potentially used as a candidate protein for screening inhibitors for virus assembly. Also, the fused protein may be used for screening aptamers (DNA, RNA, Peptide and modified aptamer) which can be used in diagnostics or therapeutics. In particular, it may be used for screening specific blockers for flavivirus entry and assembly. In addition, it may also be used for raising antibodies in mice/rabbit models which might have potential application in diagnostics and therapeutics.
In a first aspect of the invention, there is provided a fused protein comprising a moiety and a protein selected from a group consisting of flavivirus structural and non-structural (NS) proteins. Preferably, the flavivirus structural protein is a flavivirus capsid protein or a flavivirus envelope protein.
By “fused protein”, we include any protein that is produced and expressed through the joining of two of more genes which originally coded for separate proteins. It then follows that the translation of this fused gene results in a single polypeptide with functional properties derived from each of the original proteins.
Preferably, the moiety is a readily detectable moiety.
By a “readily detectable moiety”, we include the meaning that the moiety is one which, when located at the target site, for example to the target protein, may be allowed for the detection of that protein. Thus, the compounds of this embodiment of the present invention may be useful in detection and diagnosis. Preferably, the moiety is a biotin acceptor signal peptide.
Preferably, the flavivirus is a Dengue virus or a West Nile virus. Still preferably, the biotin acceptor peptide is fused with a West Nile envelope Domain III protein. Alternatively, the biotin acceptor peptide is fused at the N-terminus of the capsid protein.
Preferably, the fused protein is full-length and non-truncated.
In a second aspect of the invention, there is provided a fused protein according to the first aspect of the invention for use in medicine. Typically, the fused protein may be packaged and presented as a medicament or as a diagnostic agent for use in a patient.
In a third aspect of the invention, there is provided an isolated nucleic acid encoding the fused protein according to the first aspect of the invention.
Suitable nucleic acid molecules may readily be synthesised or constructed by the person skilled in the art using routine methods such as those described in Sambrook et al (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Typically the nucleic acid is DNA, but it may be RNA. In the following, where DNA is used, unless the context indicates to the contrary, this also includes RNA.
The nucleic acid is then expressed in a suitable host to produce a polypeptide comprising the compound of this aspect of the invention. Thus, the nucleic acid encoding the polypeptide constituting the fused protein of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
As such, in a fourth aspect of the invention, there is provided an expression vector comprising the nucleic acid according to the second aspect of the invention. Preferably, the expression vector is a plasmid.
Such techniques include those disclosed in U.S. Pat. No. 4,440,859 issued 3 Apr. 1984 to Rutter et al, U.S. Pat. No. 4,530,901 issued 23 Jul. 1985 to Weissman, U.S. Pat. No. 4,582,800 issued 15 Apr. 1986 to Crowl, U.S. Pat. No. 4,677,063 issued 30 Jun. 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued 7 Jul. 1987 to Goeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to Itakura et al, U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to Murray, U.S. Pat. No. 4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al, U.S. Pat. No. 4,766,075 issued 23 Aug. 1988 to Goeddel et al and U.S. Pat. No. 4,810,648 issued 7 Mar. 1989 to Stalker, all of which are incorporated herein by reference.
The nucleic acid encoding the polypeptide constituting the fused protein of the invention may be joined to a wide variety of other nucleic acid sequences for introduction into an appropriate host. The companion nucleic acid will depend upon the nature of the host, the manner of the introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired.
Generally, the nucleic acid is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the nucleic acid may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a nucleic acid sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
In a fifth aspect of the invention, there is provided a host cell comprising the expression vector according to the fourth aspect of the invention.
Host cells that have been transformed by the recombinant nucleic acid of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
The vectors include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
A promoter is an expression control element formed by a nucleic acid sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a nucleic acid segment of the present invention.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers his3, trp1, leu2 and ura3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
A variety of methods have been developed to operatively link nucleic acid to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the nucleic acid segment to be inserted to the vector nucleic acid. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3′-single-stranded termini with their 3′-5′-exonucleolytic activities, and fill in recessed 3′-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of this aspect of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
In a sixth aspect of the invention, there is provided an immunogenic composition or vaccine comprising a fused protein according to the first aspect of the invention.
The vaccine may advantageously contain other components, such as adjuvant, attenuated flavivirus, killed flavivirus or subunits thereof, and/or other immunogenic molecules against the flavivirus, in particular against the envelope protein of the flavivirus. The manufacturing process and components to be included in the vaccine are known as such to the person skilled in the art.
The vaccine disclosed herein may be administered by any suitable route, which delivers an immunoprotective amount of the fused protein and other immunogenic components of the vaccine to the patient. Routes of administration of the vaccine, such as, for example, parenteral route, intramuscular route or deep subcutaneous route, are identifiable by a person skilled in the art. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
A person skilled in the art can determine the appropriate immunoprotective and non-toxic dose of such vaccine to be administered. The appropriate immunoprotective and non-toxic amount of the active agents in the vaccine are in the range of the effective amounts of antigen in conventional vaccines including active agents. The specific dose level for a specific patient will be determined with reference to the age, sex, and general health of the patient. Also, the synergistic effect with other drugs administered as well as the diet of the patient, the time and route of administration, and the degree of protection to be sought, will be taken in consideration to determine the appropriate immunoprotective dose for the patient. The administration can be repeated at suitable intervals, if necessary.
In a seventh aspect of the invention, there is provided a pharmaceutical composition for treating a flavivirus infection in a patient, the composition comprising a therapeutic effective amount of a fused protein or fragment thereof according to the first aspect of the invention and at least one pharmaceutically acceptable carrier, excipient or diluent.
The carrier(s) must be “acceptable” in the sense of being compatible with the fused protein of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used.
The pharmaceutical compositions or formulations of the present invention may be for parenteral administration, for example for intravenous administration. In which case, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Alternatively, or suitably, the pharmaceutical compositions or formulations of the present invention may be for oral administration.
The exact amount required for the treatment of a patient, and route of administration of such amount will vary from patient to patient, depending on the species, age, and general condition of the individual patient, the severity of the infection, the particular antiviral agent and its mode of administration, etc.
In an eighth aspect of the invention, there is provided a method for treating a flavivirus infection in a patient, the method comprising the step of administering to the patient a therapeutically effective amount of a fused protein or fragment thereof according to the first aspect of the invention.
In a ninth aspect of the invention, there is provided a fused protein according to the first aspect of the invention, a pharmaceutical composition according to the seventh aspect of the invention, a nucleic acid according to the third aspect of the invention, or an expression vector according to the fourth aspect of the invention for use in the treatment or prevention of a flavivirus infection in a patient.
In a tenth aspect of the invention, there is provided a use of a fused protein according to the first aspect of the invention, a pharmaceutical composition according to the seventh aspect of the invention, a nucleic acid according to the third aspect of the invention, or an expression vector according to the fourth aspect of the invention in the preparation of a medicament for the treatment or prevention of a flavivirus infection in a patient.
In a eleventh aspect of the invention, there is provided a method for producing a fused protein, the method comprising: (a). obtaining a gene clone derived from a flavivirus capsid protein; (b). obtaining a biotin acceptor peptide gene; (c). producing an expression vector through the joining of the gene clone derived from the flavivirus capsid protein and the biotin acceptor peptide gene; (d). expressing the gene of the fused protein by transforming the expression vector to a host; and (e). purifying the fused protein.
Preferably, the host is Escherichia coli.
Preferably, the purifying step includes the use of urea, non-ionic detergent, ion exchange chromatography and size exclusion chromatography.
In a twelfth aspect of the invention, there is provided a fused protein produced by the method according to the eleventh aspect of the invention for use in the treatment of a flavivirus infection in a patient.
In a thirteenth aspect of the invention, there is provided a method of screening a library of molecules to identify or select one or more molecules thereof which selective bind to a fused protein, or fragment thereof, according to a first aspect of the invention, the method comprising: (a) contacting the library of molecules with the fused protein; and (b) detecting binding of one or more molecules to the fused protein.
Preferably, the method further comprises labelling the molecule with a biotin binding agent and binding is detected by detecting the labelled molecule.
Preferably, the molecule is selected from a group consisting of antibodies and aptamers.
By “antibody”, we include polyclonal or monoclonal antibody unless differently specified. The relevant preparation, is identifiable by a person skilled in the art upon reading of the present disclosure. In the specific examples given, murine polyclonal antibodies were used. Monoclonal antibodies may be obtained by any technique that provides for the production of antibody molecules by continuous cell line culture. These techniques are well known and routinely used in academic and industrial settings. Some techniques include but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alen R. Liss, Inc., pp. 77-96). Antibody fragments, which retain the ability to recognize the antigen of interest, are included as well.
The antibodies are produced using techniques known to those skilled in the art and disclosed, for example, in immunization techniques in vivo or in vitro. These techniques are well known and routinely used in academic and industrial settings.
By “aptamer”, we include any oligonucleic acid or peptide molecules that specifically target and bind to any molecule.
In a fifteenth aspect of the invention, there is provided a diagnostic kit comprising a fused protein according to the first aspect of the invention and instructions for use of the same. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).
Methods, kits, vaccine and pharmaceutical compositions disclosed herein are particularly used when the flavivirus is a member of the Japanese encephalitis serocomplex, preferably West Nile Virus, Japanese Encephalitis virus, West Valley or a virus such as Dengue and Kunjin virus. Preferably, the vertebrate is a mammal, and, in particular, a human being.
A person skilled in the art can identify modalities, dosages, timing of administration of the methods herein disclosed as well as vehicle carrier auxiliary agents, relative concentration, formulation and modalities of administration of the compositions herein disclosed.
The invention will now be described with reference to the following none limiting figures and examples.
All references herein mentioned are hereby incorporated by reference.
Supplementary
Supplementary
Supplementary Table 1. DNA (SEQ ID NO: 17) and protein (SEQ ID NO: 18) sequences of full-length DENV C protein
Supplementary Table 2. Rare codon analysis of full-length DENV C protein. Rare codons are in BOLD and underlined. The full-length DENV C DNA sequence is SEQ ID NO: 14.
Supplementary Table 3. Competent cells identified through BLAST analysis against BirA gene.
Supplementary Table 4. MALDI-TOF mass spectrometry analysis of purified DENV C protein, including FSLGMLQGR (SEQ ID NO: 15) and FLTIPPTAGILKR (SEQ ID NO: 16).
The present invention relates to a fused protein (in vivo site specific biotinylation of a viral (of the Flavivirus genus) protein), a method of producing the protein and application of the protein. The general steps for the production of fused protein—in an embodiment of the present invention, the fused protein may be a biotinylated Dengue virus Capsid protein or a West Nile virus Envelope protein DIII—are shown in
DENV C gene was amplified from cDNA synthesized from DENV-2 (NGC strain; accession number: M29095; nucleotide 100-438) RNA using SuperScript™ III First-Strand Synthesis System (Life Technologies, USA). Primers Biotin_F (5′-CTAGCTAGCTCCGGCCTGAACGAC-3′) (SEQ ID NO: 1), Biotin_C_F (5′-GACGACGACAAGAGCATGAATAACCAA-3′) (SEQ ID NO: 2), Biotin_C_R (5′-TTGGTTATTCATGCTCTTGTCGTCGTC-3′) (SEQ ID NO: 3), and C_R (5′-CCGCTCGAGTTACGCCATCACTGT-3′) (SEQ ID NO: 4) were used to join biotin acceptor peptide gene (Cull and Schatz, 2000) containing an enterokinase cleavage site with DENV C gene via overlap extension PCR (OE-PCR). Gel-purified PCR products containing the joined fragments were subsequently inserted into expression vector, pET28a (Novagen, Germany) via NheI and XhoI cut sites. 6×His tag and thrombin cleavage site are at the N-terminus of signal peptide followed by enterokinase cleavage site and DENV C protein. DNA sequences of the constructs were confirmed by sequencing.
Transformed bacterial colonies of each strain [BL-21 (DE3) and BL-21-CodonPlus] (Agilent Technologies, USA) were picked and grown in 20 ml Luria-Bertani (LB) broth with 30 μg/ml kanamycin antibiotic. When the bacteria absorbance OD600nm reached 0.65, protein expression was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) overnight at 28° C. After IPTG induction, the bacteria absorbance OD600nm was measured again and 200 μl of equal bacteria density for both strains were used for expression level screening. Bacterial cells were pelleted down with centrifugation at 8,000 rpm for 15 min at 4° C. and resuspended with 100 μl 1×protein sample buffer containing β-mercaptoethanol. The samples were boiled for 10 min with constant vortexing at 1 min interval. Insoluble substances were pelleted down with centrifugation for 2 min at 20,000 ×g.
pET28aDENVBioCap plasmid was transformed into BL-21-CodonPlus expression competent cells (Agilent Technologies, USA) and grown on LB agar containing 30 μg/ml kanamycin. Selected clones were cultured in 1 L LB broth (30 μg/ml kanamycin) at 30° C. until absorbance OD600nm of 0.65. Expression of DENV C protein was induced with 1 mM IPTG overnight at 28° C. Bacterial cells were pelleted down with centrifugation at 8,000 rpm for 15 min at 4° C. Pellet was then resuspended in 10 ml resuspension buffer (20 mM Tris, 300 mM NaCl, 0.2% Triton-X, pH 8.0) and pelleted down again at 8,000 rpm for 15 min. The pellet was washed with wash buffer (20 mM Tris, 300 mM NaCl, pH 8.0) before it was resuspended in 30 ml lysis buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 10 mM Imidazole, pH 8.0) containing EDTA-free protease inhibitor (Roche, Switzerland). The mixture was incubated at room temperature for 30 min and the lysate was subsequently clarified by centrifugation at 13,200 rpm for 20 min.
Bacterial lysate containing denatured DENV C protein was incubated with nickel-nitrilotriacetic acid (Ni-NTA) resin (Bio-Rad, USA) for binding in a chromatography column overnight at 4° C. Ten column volume of wash buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 20 mM Imidazole, pH 8.0) was used to wash away non-specific binding proteins. DENV C protein was eventually eluted out with elution buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) in ten fractions. Next, all eluates were combined for refolding and dialysis to remove 8 M urea. Briefly, all eluates were pooled into a SnakeSkin dialysis membrane tubing, 3.5 k MWCO, (Thermo Scientific, USA) and 0.5% of Tween-20 was added into the samples. The dialysis tubing was incubated in 1 L of 6 M urea for 6-12 hr at 4° C. and 250 ml of 25 mM Tris (pH 8.0) was added into the solution at every 6-12 hr interval. When the final volume reached 3 L, the dialysis tubing was transferred into 2 L of 20 mM Tris (pH 8.0) for 6 hr. Refolded DENV C proteins were collected from the dialysis tubing.
Ion exchange chromatography column, Resource™ Q/Z 1 ml-packed size column (GE Healthcare, UK), was connected to fast protein liquid chromatography (FPLC) system. The column was equilibrated with ten column volumes of 20 mM Tris (pH 8.0) until the UV baseline and conductivity are stable. Refolded DENV C protein was injected into the column and the flow rate was set to 0.5 ml/min. After the sample passed through the column, ten column volumes of 20 mM Tris (pH 8.0) was used to wash away all the unbound proteins. Ionically-bound proteins were eluted out with increasing concentration of sodium chloride to a final concentration of 1 M (100%). All the fractions were analyzed via ELISA to determine the presence of biotinylated C protein.
Superdex 75 10/300 GL chromatographic separation column (GE Healthcare, UK) was connected to FPLC system. The column was washed with five column volumes of MilliQ™ water and then equilibrated with two column volumes of 1× phosphate buffered saline (PBS) (pH 7.2). Samples were injected into the column and the flow rate was set to 0.25 ml/min. All the eluates were analyzed via ELISA to determine the presence of biotinylated C protein.
To determine whether the expressed protein is biotinylated or not, 50 μl of samples were added into the wells of MaxiSorp plate (eBioscience, USA) in triplicate for coating overnight at 4° C. After washing with 1×0.1% PBST, 150 μl blocking buffer (4% bovine serum albumin) was added into each well and incubated for another hour at room temperature. Next, 150 μl streptavidin-HRP enzyme conjugates (1:5000 dilution) was added and incubated for 1 hr. The plate was washed with 1×PBST three times to remove unbound conjugates and then 100 μl substrate solution, tetramethyl benzidine [(TMB) (Promega, USA)], was added for development. To stop the reaction, 50 μl of 0.5 M H2SO4 solution was added. The absorbance was measured at 450 nm.
Samples collected from flow through, wash, and eluates were analyzed by SDS-PAGE and Western blot. Twelve % Tris-tricine polyacrylamide denaturing gel was used to separate proteins in the samples and subsequently stained with Coomassie blue for detection. The presence of biotinylated DENV C protein was confirmed by Western blot via two different approaches. First, the identity of DENV C protein was determined with anti-His antibody. Briefly, separated proteins were transferred from polyacrylamide gel onto a PVDF membrane using iBlot® Dry Blotting System (Life Technologies, USA). Blocking was done with 5% skimmed milk for 1 hr at room temperature. Next, the membrane was incubated with 0.1 μg/ml mouse anti-His antibody (Qiagen, Germany) overnight at 4° C. The membrane was then washed with 1×TBST and incubated with 0.1 μg/ml goat anti-mouse secondary antibody conjugated with HRP (Thermo Scientific, USA) for 1 hr at room temperature. After washing with 1×TBST, the membrane was developed using SuperSignal® West Pico/Dura/Femto chemiluminescent substrate (Thermo Scientific, USA).
For the second approach, DENV C protein was detected directly using streptavidin conjugated with HRP. After transferring the samples onto a PVDF membrane, it was blocked with 4% bovine serum albumin (BSA) for 1 hr at room temperature. The membrane was then incubated with HRP-conjugated streptavidin (Millipore, USA) for another hour at room temperature. Subsequently, the membrane was washed thoroughly with 1×PBST for 1 hr at room temperature and developed with chemiluminescent substrate.
Purified protein was electrophoresed through SDS-PAGE using 12% Tris-tricine polyacrylamide denaturing gel and stained with Coomassie blue. The background of Coomassie-stained gel was removed with destaining solution (40% methanol, 10% glacial acetic acid, 50% distilled H2O). DENV C protein-corresponding band was excised from the gel and kept in eppendorf tube containing distilled water. Samples were submitted to Dr. Lim Yong Pin (Department of Biochemistry, NUS) for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis.
The binding affinity of purified biotinylated DENV C protein was tested using streptavidin magnetic beads (GE Healthcare, UK) according to the manufacturer's protocol. Briefly, samples were mixed with the streptavidin magnetic beads and incubated for 1 hr with gentle mixing at 4° C. Unbound proteins were removed with wash buffer while biotinylated proteins were eluted out with elution buffer provided in the kit. Eluted proteins were analyzed by ELISA to confirm the biotinylation.
Pure Sec3 protein [(Abnova, Taiwan) (known interacting partner of flavivirus C protein (Bhuvanakantham, Li, Tan and Ng, 2010))] was added into the wells of MaxiSorp plate (eBioscience, USA) for coating overnight at 4° C. After washing with 1×PBST, blocking buffer (4% bovine serum albumin) was added into each well and incubated for another hour. Purified DENV C protein was added into the well for binding at 37° C. for 1 hr. Next, streptavidin-HRP enzyme conjugates was added and incubated for another hour at 37° C. After washing three times with 1×PBS, 100 μl TMB substrate (Promega, USA) was added for development and 50 μl 0.5 M H2SO4 solution was added to stop the reaction when necessary. The absorbance was measured at 450 nm.
To avoid non-specific biotinylation, we engineered a biotin acceptor peptide (BAP) at the N-terminus of DENV C protein. Biotin is a very small molecule (molecular weight =244.31) so it is unlikely to affect the structure and function of the protein. To ensure that the BAP will not result in any steric hindrance to the protein conformation, a secondary structure prediction was performed. As shown in
To clone the full-length DENV C protein, nucleotides 100 to 438 from DENV-2 genome (GeneBank accession number: M29095) was amplified (Fragment B in
The final ligated product was then cloned into bacterial expression vector, pET28 which consisted of a 6×His tag and a thrombin cleavage site in the upstream of the multiple cloning sites. Hence, the recombinant full-length DENV C construct (pET28aDENVBioCap) contained 2 tags (6×His tag and BAP) at the N-terminus and 2 different enzyme cleavage sites (thrombin cleavage site and enterokinase cleavage site) for tag removal when necessary.
Five successfully transformed bacterial colonies were picked for colony PCR screening and DNA sequencing was performed to verify the constructs. The final DNA and protein sequence were shown in Suppl. Table 1. To further support our site-specific biotinylation strategy, WNV domain III (DIII) protein which has similar molecular weight as the C protein, was also engineered with a BAP concurrently via the same procedure. DENV C and WNV DIII proteins without BAP were also constructed for comparison purposes.
To express DENV C protein, an optimal expression competent bacterial strain is requisite. We detected 14 rare codons in the full-length DENV C protein DNA sequence (Suppl. Table 2). This raised a concern for protein expression because 12% of the total 115 codons are rare codons. To screen for the optimal bacterial expression competent cell, pET28aDENVBioCap plasmid was transformed into BL-21 (DE3) and BL-21-CodonPlus. BL-21 (DE3) is a common bacterial strain for high expression of recombinant protein while BL-21-CodonPlus is a bacterial strain specifically engineered for expression of protein with rare codons.
The expression of DENV C protein was indeed much higher in BL-21-CodonPlus strain. As indicated in
As comparing DENV C protein with and without BAP via Western-blot using anti-His antibody, bands were observed in all the lanes for DENV C and WNV DIII proteins (
Besides detecting the proteins of interest using anti-His antibody, streptavidin-horseradish peroxidase (HRP) was also employed to detect the presence of biotinylated protein. Surprisingly, we found that our proteins of interest were biotinylated even before any in vitro biotinylation process. As shown in FIG. 3Bii, thick bands can be observed in the BAP-containing DENV C and WNV DIII proteins when streptavidin-HRP antibody was used. Similar bands were not detected in DENV C and WNV DIII proteins without BAP. Commercially-available biotinylated (BN) and unbiotinylated (UBN) maltose-binding protein [(MBP) (GeneCopoeia, USA)] were used as positive and negative controls for biotinylation, respectively. Similarly, band was only seen in the biotinylated MBP Lane. This result suggested that DENV C and WNV DIII proteins with BAP were biotinylated endogenously in BL-21 strains.
We postulated that bacterial BL-21 strains may contain biotin holoenzyme synthetase BirA gene that encodes for biotin ligase protein in their genome. To unveil the mystery as to why biotinylation could occur endogenously, BirA gene sequence was analyzed using BLAST software and the blasting result showed that BL-21 strains indeed possess BirA gene in their genome (Supplementary Table 3). It was also reported that biotin molecules were present in the Luria-Bertani (LB) broth (Tolaymat and Mock, 1989). As a result, proteins engineered with BAP could be directly expressed and biotinylated in BL-21 strains without the extra in vitro enzymatic biotinylation step.
After confirming the expression and biotinylation of the protein of interest, large scale production of bacterial culture was carried out. Initial attempt was to perform all the extraction and purification steps in native form so that the protein structure could be preserved without the need of refolding. However, the extraction of DENV C protein from the bacterial cells was not effective in native condition. There were still considerably huge amount of DENV C protein trapped in the pellet (data not shown). Therefore, 8 M urea was used to lyze the bacterial cells and affinity his-tag chromatography purification was first performed under denaturing condition.
The bacterial cell lysate with biotinylated DENV C protein was incubated with nickel-nitrilotriacetic acid (Ni-NTA) resin to purify His-tagged proteins. Recombinant full-length DENV C protein contained 6×His tag at the N-terminus bound to the resin and unbound proteins were removed during washing with 20 mM imidazole. To ensure most of the unbound proteins were washed away, ten column volumes of wash buffer were used. The bound proteins were eluted out with 500 mM imidazole. However, many non-specific bands were still observed in the eluates of DENV C protein (
Partially purified DENV C proteins from eluates E2 to E10 were pooled together for step-wise dialysis and concurrent refolding. Due to the hydrophobic C-terminus of DENV C protein, the protein easily aggregated resulting in major loss during refolding and dialysis. To prevent protein aggregation and to enhance efficient refolding, 0.05% of Tween-20 was added to the samples (Krupakar et al., 2012). With this addition of detergent in the samples, protein aggregation was reduced significantly. No obvious precipitation was observed and the solution was clear after dialysis and refolding. This partially purified DENV C protein was then subjected to second round of purification using ion exchange chromatography-fast protein liquid chromatography system (IEX-FPLC).
Theoretically, DENV C protein is a positively-charged protein so cation-exchanger column, Resource MonoS, should be used. However, we found that biotinylated DENV C protein was not eluted out at a specific concentration of sodium chloride (NaCl). Miniature amount of the protein was eluted out slowly in an increasing gradient manner and it reached plateau at 70 mM of NaCl (Suppl.
Counter intuitively, we were able to obtain better separation when we used anion-exchanger, Resource MonoQ, column (
To avoid any other contaminants that may have similar charge as biotinylated DENV C protein, eluates corresponding to the high UV absorbance peak after IEX-FPLC were injected into size-exclusion chromatography (SEC), Superdex 75 10/300 GL column. It was previously reported that SEC could not be used to estimate the molecular weight of DENV C protein because there were many unspecific interaction between the gel matrix and C protein (Jones, Ma, Burgner, Groesch, Post and Kuhn, 2003). Nevertheless, it is still a good method to further isolate the proteins of interest from other possible residual contaminants based on the molecular weight differences. After size-exclusion chromatography, three peaks were observed in the chromatogram (
With this optimized sequential purification protocol, we were able to obtain 0.8-1.0 mg of highly purified biotinylated non-truncated, full-length DENV C protein from 1 L of IPTG-induced bacterial culture repeatedly.
To compare the biotinylation efficiency of our strategy, the same amount of purified biotinylated DENV C, WNV DIII and MBP proteins were coated on the ELISA plate and streptavidin-HRP antibody was used for detection. Assuming that the coating efficiencies are the same among all three proteins, the absorbance detected via direct ELISA should be proportional to the number of coated proteins and inversely proportional to the power of ⅔ of its molecular size. The larger the protein, the lesser the proteins can be coated on the plate.
The molecular size of biotinylated MBP is about two times larger than biotinylated DENV C and WNV DIII proteins. Assuming that one protein only carries one biotin which will bind to one streptavidin-HRP only, the absorbance of MBP should be two times lower than our purified proteins for the same protein concentration. As shown in
This result further supported that engineering an additional BAP on a protein could result in site-specific endogenous biotinylation with high efficiency in Escherichia coli BL-21 strains, without the need of an extra in vitro enzymatic or chemical conjugation step. Our strategy could produce biotinylated proteins that were as good quality as the manufactured biotinylated MBP.
It is essential to ensure that the purified proteins are functional before they are used for any further studies. According to our recent findings, WNV and DENV C proteins were found to interact with human Sec3 exocyst protein (Bhuvanakantham, Li, Tan and Ng, 2010). Thus, we examined the functionality of the purified DENV C protein by revisiting the interaction between biotinylated DENV C protein and Sec3 protein using ELISA. Pure Sec3 protein was coated overnight on ELISA plate and purified biotinylated DENV C protein was used as the probe. Bound DENV C proteins were detected with streptavidin-HRP antibody. As shown in
Dengue virus (DENV) is a positive-stranded RNA virus. Based on the current understanding of flaviviral replication cycle, host cell nucleus is not involved during the transcription, translation and assembly of virus. This traditional notion is challenged when capsid (C) protein is found to localize in the infected cell nucleus. Since then, the role of DENV C protein in the nucleus has been an intriguing mystery for researchers to unveil. Understanding the roles of C protein in the nucleus will not only completes the missing puzzle of flaviviral replication cycle, but also provides insights for novel anti-viral strategies development.
Using the purified biotinylated full-length DENV C protein of the present invention, high-throughput screening (HTS) of novel interacting partners of DENV C protein is made possible. ProtoArray® Human Protein Microarray PPI Complete Kit (Life Technologies, USA) was employed for HTS study of novel DENV C protein-interacting partners. This protein microarray contains 9400 purified human proteins printed in duplicate on the nitrocellulose-coated glass slide. The purified human proteins encompass various cellular kinases, enzymes, nuclear proteins and signalling proteins tagged with glutathione S transferase (GST) or 6×His. Purified biotinylated DENV C protein was used as probe and streptavidin-Alexa Fluor® 647 antibody is chosen as the detection reagent as it yields very good signal to noise ratio.
There were 578 potential DENV C protein-binding partners detected to have z-score value above 1 (
Z-score value of 3 was chosen to be the cut-off point for statistically significant potential interactors. Z-score indicates how far and in what direction the sample's value deviates from the distribution's mean. After filtering some of the proteins without known functions and with biotin-binding property, 31 significant potential interacting partners were identified for DENV C protein as shown in the table below.
Scanned images of the microarray are analyzed using GenePix® Pro software to determine the pixel intensity and the data acquired are further analyzed via Protoarray® Prospector software to identify potential interacting partners of DENV C protein. Z-score cut-off value of 3 is chosen to be the statistically significant interactors.
S.
cerevisiae)
The list of potential interacting partners for DENV C protein was quite well dispersed throughout the cell nucleus and cytoplasm. The total proteins in each cellular compartment are calculated and charted.
From the list of potential interacting partners, it appeared that DENV C protein may be involved primarily in cell cycle control and regulation of transcriptional and translational activities (
The purified biotinylated full-length Dengue virus capsid protein of the present invention can also be used to verify the binding between DENV C protein and its interacting partners in ELISA platform. To validate the identified potential interacting partners from the protein microarray study, commercially-available pure proteins were purchased and coated on ELISA plate. Biotinylated full-length DENV C protein was used as probe for binding and streptavidin-HRP antibody was used to detect the bound proteins. Four proteins (DIMT1, EIF1AX, EIF1AY, and SRP19) that are related to transcriptional and translational machinery and five proteins pertaining to cell cycle control (UBE2S, CARF, CHES1, CCNB3, and VRK1) were selected for further validation of DENV C protein binding. As shown in
Therefore, the biotinylated full-length DENV C protein of the present invention is useful to flavivirus laboratories that are studying protein-protein, protein-RNA, and protein-DNA interactions.
One of the challenges in Dengue virus research is that antibodies against the viral proteins are not available commercially. Most of the laboratories need to raise the antibodies on their own before carrying out any further downstream experiments (Puttikhunt et al., 2009, Wang et al., 2002). Anti-DENV C antibody is one of the missing antibodies in the market. Hence both full-length DENV C protein and anti-DENV C antibody are of commercial values.
Before the present inventors raised anti-C antibody using pure DENV C protein, an antibody generation package from Abmart Inc. was purchased to produce monoclonal antibodies against DENV C peptides. Abmart Inc. was only able to produce monoclonal antibodies against two peptides (MNNQRKKARN (SEQ ID NO: 5) and ERNRVSTVQQ (SEQ ID NO: 7)). Below is a list of predicted epitopes for antibody production:
The epitopes are predicted by Abmart Inc. using their propriety algorithm. An “epitope score” is assigned to each residue based on criteria such as structural features, sequence conservation, hydrophobicity, and solvent exposure. Nine 10-residue fragments with highest overall scores are chosen as epitopes to generate monoclonal antibodies.
The first peptide (MNNQRKKARN) (SEQ ID NO: 5) is the first ten residues from the N-terminus of C protein whereas the second peptide (ERNRVSTVQQ) (SEQ ID NO: 7) is from the 19th residue to the 28th residue. Both epitopes are from the N-terminal region before the first helix (α1) structure. Monoclonal antibodies against these two peptides were tested to be able to recognize purified full-length recombinant DENV C protein as shown in
To produce biotinylated proteins, we opted for site-specific biotinylation by engineering a biotin acceptor signal peptide (BAP) in the upstream of our proteins. To attach a biotin molecule onto BAP either in vivo or in vitro, bacterial biotin ligase BirA enzyme is required (Chapman-Smith and Cronan, 1999, Cull and Schatz, 2000, Tan et al., 2004, Yang et al., 2004). Instead of performing this biotinylation step in vitro, we found that biotinylation of the BAP-containing proteins occurred endogenously in Escherichia coli BL-21 strains with high efficiency.
The purification of non-truncated, full-length DENV C protein was indeed challenging. Unlike DIII protein (Tan et al., 2010), one single purification step was simply not adequate to produce pure DENV C protein. The presence of rare codons in the C protein sequence necessitates unique expression competent strain for its optimal protein expression. Due to the hydrophobic C-terminus, aggregation easily occurred and most of the expressed proteins were trapped in the inclusion bodies. As such, we had to use 8 M urea to denature the proteins and perform affinity chromatography under denaturing condition. We also managed to solve the aggregation problem during dialysis and refolding by using non-ionic detergent. To obtain highly purified DENV C protein, two more purification steps were included in our protocol, namely IEX and SEC. This sequential purification strategy can produce approximately 1 mg of purified DENV C protein from 1 L of bacterial culture.
This purified non-truncated, full-length DENV C protein is useful for various molecular and structural studies. For instance, it can be used to study the interaction of NS2B-NS3 serine proteinase and C protein. In order to produce mature C protein for encapsidation and assembly, full-length C protein has to be cleaved by NS2B-NS3 protein complex. Thus, it is crucial to identify the binding sides of NS2B-NS3 complex to full-length C protein, which is still unknown. It will in turn lead to the development of novel anti-viral drugs targeting the assembly of virus particles. Besides, it is also interesting to examine whether the cleaved C-terminus plays any unidentified role in the pathogenesis. The crystal structure of DENV NS2B-NS3 protein complex has been resolved (Erbel et al., 2006). However, the crystal structure of full-length DENV C protein is still not available yet, although the NMR structure of partial DENV C protein was obtained (Ma, Jones, Groesch, Kuhn and Post, 2004). We are now taking up the challenge to resolve the 3D structure of full-length DENV C protein using our purified protein.
Ma and co-workers (2004) proposed a model for the interaction between C protein and its viral genome using the partial DENV C protein. However, this model is still not validated and the exact mechanism of encapsidation is still unclear until to date. Investigation of how DENV C protein interacts with its RNA and oligomerizes into nucleocapsid during maturation is vital for better understanding of the pathogenesis. Recent publications also demonstrated that N-terminus of DENV C protein was antigenic in mice study and the first 18 amino acid residues were involved in the virus assembly during maturation (Puttikhunt, Ong-Ajchaowlerd, Prommool, Sangiambut, Netsawang, Limjindaporn, Malasit and Kasinrerk, 2009, Samsa, Mondotte, Caramelo and Gamarnik, 2012). As a result, our purified non-truncated DENV C protein is absolutely desirable for delineating the whole process of encapsidation and virus assembly.
Another difficulty of studying DENV C protein is that anti-C antibody is yet to be available commercially. Most of the laboratories need to raise this antibody on their own before any further downstream experiments (Puttikhunt, Ong-Ajchaowlerd, Prommool, Sangiambut, Netsawang, Limjindaporn, Malasit and Kasinrerk, 2009, Wang et al., 2002). Hence, pure full-length DENV C protein can be obtained simply by enterokinase cleavage and subsequently used to raise antibody in mice or rabbit for laboratory research usage and diagnostic tool. Both full-length DENV C protein and anti-DENV C antibody will be of commercial values.
In conclusion, we have developed an optimized protocol to engineer, express and purify the first biotinylated, full-length DENV C protein. This purified protein is useful for various molecular studies to further understand the underlying mechanism of C protein in pathogenesis. Only by stitching all these missing pieces of the puzzle together, novel antiviral strategies can be rationally designed.
Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.
All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
For the purposes of this specification, it will be clearly understood that the word comprising means including but not limited to, and that the word comprises has a corresponding meaning.
1. Amberg S. M. and Rice C. M. (1999) J Virol, 73, 8083-8094.
2. Bayer E. A. and Wilchek M. (1990) Methods Enzymol, 184, 174-187.
3. Bayer E. A. and Wilchek M. (1990) Methods Enzymol, 184, 138-160.
4. Bhuvanakantham R., Chong M. K. and Ng M. L. (2009) Biochem Bioph Res Co, 389, 63-69. Bhuvanakantham R., Li J., Tan T. T. T. and Ng M. L. (2010) Cellular Microbiology, 12, 453-472.
5. Chapman-Smith A. and Cronan J. E., Jr. (1999) Trends Biochem Sci, 24, 359-363.
6. Chapman-Smith A. and Cronan J. E., Jr. (1999) Biomol Eng, 16, 119-125.
7. Cull M. G. and Schatz P. J. (2000) Methods Enzymol, 326, 430-440.
8. de Boer E., Rodriguez P., Bonte E., Krijgsveld J., Katsantoni E., Heck A., Grosveld F. And Strouboulis J. (2003) Proc Natl Acad Sci USA, 100, 7480-7485.
9. Erbel P., Schiering N., D'Arcy A., Renatus M., Kroemer M., Lim S. P., Yin Z., Keller T. H., Vasudevan S. G. and Hommel U. (2006) Nat Struct Mol Biol, 13, 372-373.
10. Gubler D. J. (2002) Arch Med Res, 33, 330-342.
11. Hase T., Summers P. L., Eckels K. H. and Baze W. B. (1987) Archives of Virology, 92, 273-291.
12. He Y. F., Bao H. M., Xiao X. F., Zuo S., Du R. Y., Tang S. W., Yang P. Y. and Chen X. (2009) Proteomics, 9, 5414-5424.
13. Howarth M. and Ting A. Y. (2008) Nat Protoc, 3, 534-545.
14. Jones C. T., Ma L. X., Burgner J. W., Groesch T. D., Post C. B. and Kuhn R. J. (2003) J Virol, 77, 7143-7149.
15. Krupakar P., Ngo A. M. L. and Ng M. L. (2012) In Miguel Benitez and Aguirre V. (eds) Protein Purification. Nova Science, New York, pp. 147-170.
16. Ma L., Jones C. T., Groesch T. D., Kuhn R. J. and Post C. B. (2004) Proc Natl Acad Sci USA, 101, 3414-3419.
17. Markham K., Bai Y. and Schmitt-Ulms G. (2007) Anal Bioanal Chem, 389, 461-473.
18. Markoff L. (1989) J Virol, 63, 3345-3352.
19. Moreland N. J., Tay M. Y., Lim E., Paradkar P. N., Doan D. N., Yau Y. H., Geifman Shochat S. and Vasudevan S. G. (2010) PLoS Negl Trop Dis, 4, e881.
20. Postel A., Letzel T., Muller F., Ehricht R., Pourquier P., Dauber M., Grund C., Beer M. and Harder T. C. (2011) Anal Biochem, 411, 22-31.
21. Puttikhunt C., Ong-Ajchaowlerd P., Prommool T., Sangiambut S., Netsawang J.,
22. Limjindaporn T., Malasit P. and Kasinrerk W. (2009) Arch Virol, 154, 1211-1221
23. Qi Y. and Katagiri F. (2011) Methods Mol Biol, 712, 21-30.
24. Samsa M. M., Mondotte J. A., Caramelo J. J. and Gamarnik A. V. (2012) J Virol, 86, 1046-1058.
25. Sueda S., Yoneda S. and Hayashi H. (2011) Chembiochem, 12, 1367-1375.
26. Tadano M., Makino Y., Fukunaga T., Okuno Y. and Fukai K. (1989) J Gen Virol, 70 (Pt 6), 1409-1415.
27. Tan L. C., Chua A. J., Goh L. S., Pua S. M., Cheong Y. K. and Ng M. L. (2010) Protein Expr Purif, 74, 129-137.
28. Tan L. P., Lue R. Y., Chen G. Y. and Yao S. Q. (2004) Bioorg Med Chem Left, 14, 6067-6070.
29. Tolaymat N. and Mock D. M. (1989) J Nutr, 119, 1357-1360.
30. Wang S. H., Syu W. J., Huang K. J., Lei H. Y., Yao C. W., King C. C. and Hu S. T. (2002) J Gen Virol, 83, 3093-3102.
31. Yang J., Jaramillo A., Shi R., Kwok W. W. and Mohanakumar T. (2004) Hum Immunol, 65, 692-699.
32. Yang M. R., Lee S. R., Oh W., Lee E. W., Yeh J. Y., Nah J. J., Joo Y. S., Shin J., Lee H. W., Pyo S. et al. (2008) Cell Microbiol, 10, 165-176.
33. Zhang W., Chipman P. R., Corver J., Johnson P. R., Zhang Y., Mukhopadhyay S., Baker T. S., Strauss J. H., Rossmann M. G. and Kuhn R. J. (2003) Nat Struct Biol, 10, 907-912.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201208602-1 | Nov 2012 | SG | national |
This application is a divisional of U.S. application Ser. No. 14/646,845, filed May 22, 2015, which is a U.S. National Phase of International Patent Application No. PCT/SG2013/000501, filed Nov. 22, 2013, which claims the benefit of Singapore Application No. 201208602-1, filed Nov. 22, 2012, each of which is incorporated by reference in its entirety herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14646845 | May 2015 | US |
| Child | 15376917 | US |
