Fusion protein comprising Gaussia luciferase, translation interrupter sequence, and interferon amino acid sequences
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10435695-B2
Publication Date
2019-10-08
Expiration Date
2036-09-08
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Abstract
Polynucleotides encoding fusion proteins comprising interferons and luciferases which have biotherapeutic, diagnostic, and quality control applications in biotechnological, medical, and veterinary fields.
Core Innovation
The invention discloses fusion proteins comprising Gaussia luciferase (GLuc) or super-luminescent Gaussia luciferase (SGLuc) reporter sequences fused to interferons. These fusion proteins are encoded by polynucleotides that may optionally include translation interrupter sequences such as Δ1D2A, allowing the expression of either fused proteins or separate proteins upon translation. This design facilitates secretion and maintains luciferase activity, enabling effective quantification of interferons or other biologically active molecules.
The background identifies the issue that existing methods for quantifying interferons, such as activity assays or antibody-based ELISA, are either indirectly related or inaccurate due to antibody cross-reactivity or variable binding affinities. These drawbacks raise challenges in reliably determining absolute concentrations of interferons. To solve this, the inventors developed chimeric proteins that combine luciferase reporters with interferons, allowing direct and straightforward measurement of interferon concentration through luminescence without depending on antibodies.
The inventors provide multiple embodiments of fusion constructs, including fusion proteins where luciferase and interferon sequences are fused directly or separated by a translation interrupter sequence. Constructs such as SGLucON α, which fuse SGLuc directly with the activity domain of IFN α, enable secretion of intact fusion proteins. Quantification of luminescence is correlated with interferon concentration, providing a simple, fast, and reliable method for determining absolute interferon concentrations. This approach addresses the problems of antibody-based detection and indirect activity assays by offering a luminescence-based direct quantification.
Claims Coverage
The patent includes one independent claim addressing the polynucleotide encoding of fusion proteins comprising luciferase and interferon sequences. The main inventive features involve fusion protein composition, inclusion of translational interrupter sequences, vector incorporation, host cell expression, and methods for quantification and application of the fusion proteins.
Polynucleotide encoding fusion proteins with luciferase and interferon
A polynucleotide that encodes a fusion protein comprising a luciferase and at least one interferon, wherein the polynucleotide encodes specific sequences as identified by SEQ ID NOS: 48, 52, 56, 60, 64, 68, 70, 74, 78, 82, 86, 90, 94, 111, or 113.
Inclusion of translational interrupter sequences
The polynucleotide or vector may further comprise polynucleotide sequences encoding one or more translational interrupter sequences such as 2A, Δ1D2A, Aphthovirus 2A, or foot-and-mouth disease virus (FMDV) 2A sequences to enable expression of separate polypeptides from a single open reading frame.
Vectors comprising fusion protein-encoding polynucleotides
Vectors comprising the polynucleotide that encodes the fusion protein along with regulatory elements like promoters or translation initiation sequences, capable of expressing the fusion protein in various host cells including mammalian, insect, or prokaryotic cells.
Host cells expressing fusion proteins
Host cells comprising vectors coding for the fusion protein, which express the fusion protein or its fragments, enabling production and secretion of these fusion proteins or separated components.
Fusion proteins encoded by the polynucleotide
Fusion proteins comprising a luciferase (GLuc or SGLuc), optionally fused or separated by translational interrupter sequences, with an interferon, which retain bioluminescent and biological activity properties.
Methods for quantifying interferon expression
Methods for quantifying the amount or concentration of interferon produced in an expression system by transforming host cells with vectors encoding the fusion protein, culturing the cells, harvesting the medium, and measuring luminescent intensity as a proxy for interferon levels.
Methods for facilitating secretion and recovery
Methods to facilitate secretion of the fusion protein from host cells and recovering the secretable fusion proteins from culture medium, enabling production and isolation for further use.
Methods for measuring biotherapeutic peptides in subjects
Methods for measuring amounts of biotherapeutic peptides comprising luciferase-fusion proteins in biological samples from subjects transformed with vectors encoding the fusion protein, detecting luminescence to determine expression levels.
Methods for certifying vaccine expression in vivo
Methods to certify in vivo expression of vaccine peptides fused to luciferase in host organisms by recovering biological samples and measuring luminescent output, confirming expression of the vaccine polypeptides.
Pharmaceutical compositions and biotherapeutics
Compositions comprising the fusion proteins and pharmaceutically acceptable carriers, adjuvants, or excipients for therapeutic application, including treatment of viral infections and immunotherapy.
The independent claims focus on polynucleotides encoding fusion proteins that combine luciferase and interferons, optionally including translational interrupters, incorporated into vectors and host cells, along with methods for expression, secretion, quantification via luminescence, therapeutic use, and vaccine certification. This coverage protects the composition, production, application, and methods related to luciferase-interferon fusion proteins.
Stated Advantages
Provides a simple and fast method for substantially quantifying absolute concentrations of interferons or biologically active molecules by measuring luminescence without the drawbacks of antibody-based detection.
Avoids inaccuracies and high costs associated with antibodies used in ELISA assays, overcoming variability in antibody binding affinities and limited shelf life.
Enables direct quantification of interferon concentration when using fusion proteins without translational interrupters, reducing biases related to differential degradation or secretion of separate protein components.
Fusion proteins retain both luciferase and biological activities, including antiviral properties, supporting therapeutic applications.
Documented Applications
Biotherapeutic use in medical and veterinary fields, including treatment of diseases such as hairy cell leukemia, malignant melanoma, AIDS-related Kaposi's sarcoma, multiple sclerosis, feline and canine viral infections, and Foot-and-Mouth Disease Virus (FMDV) in livestock and cattle.
Diagnostic and quality control applications for accurate measurement and standardization of interferon and other biologically active molecules in expression systems.
Pharmaceutical compositions comprising the fusion proteins for cytokine therapy, antiviral therapy, and vaccine development.
Methods for certifying vaccine expression in vivo by detecting luminescent signals from fusion proteins in biological samples.
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