Fusion proteins containing luciferase and a polypeptide of interest
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10829770-B2
Publication Date
2020-11-10
Expiration Date
2036-09-08
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Polynucleotides encoding fusion proteins contain a secretable luciferase fused to a modified polypeptide of interest are disclosed. The polypeptide of interest has been modified to remove a native N-terminal secretion sequence and has been replaced by the secretable luciferase. One example of a modified polypeptide of interest is interferon. The polynucleotides and fusion proteins have biotherapeutic, diagnostic, and quality control applications in biotechnological, medical, and veterinary fields. Methods for producing the secretable fusion protein are also disclosed.
Core Innovation
The invention discloses polynucleotides encoding fusion proteins comprising a secretable luciferase fused to a modified polypeptide of interest, where the polypeptide has its native N-terminal secretion sequence removed and replaced by the secretable luciferase. One exemplary modified polypeptide is interferon, which is fused to Gaussia Luciferase (GLuc) or Super-luminescent Gaussia Luciferase (SGLuc). This design facilitates secretion and activity of the fusion proteins, enabling their use in biotherapeutic, diagnostic, and quality control applications in medical and veterinary fields.
The problem addressed by the invention arises from limitations in existing methods of quantifying interferon concentrations, which typically rely on activity assays or antibody-based methods such as ELISA. Activity assays are only indirectly related to interferon concentration and may be affected by enhancements from other molecules, yielding inaccurate quantification. Antibody assays can be unreliable due to variable antibody affinities and cross-reactivities, plus high production costs and limited shelf life. Furthermore, natural interferons have secretion domains that are not essential for protective activity. Therefore, there was a need for novel chimeric proteins that enable easy and accurate determination of absolute concentrations of interferons and other biologically active molecules.
The invention solves this by creating fusion proteins that combine a secretable luciferase with an interferon or polypeptide of interest where the secretion peptide of the polypeptide is replaced by the luciferase. These fusion proteins can be expressed and secreted from host cells in a form that retains biological activity. The luciferase activity provides a direct or correlative luminescent measure of polypeptide concentration, avoiding the pitfalls of indirect or antibody-based assays. Constructs may include translational interrupter sequences (such as Aphthovirus 2A or Δ1D2A) enabling expression of separate polypeptides or a direct fusion protein without interruption for direct quantification.
Claims Coverage
The claims cover multiple independent claims that focus on polynucleotides encoding fusion proteins, the fusion proteins themselves, and methods of production and quantification. There are two main independent claim groups: those involving fusion proteins with translational interrupter sequences and those involving fusion proteins with luciferase fused directly to a modified polypeptide of interest replacing the native secretion domain.
Polynucleotide encoding a fusion protein with luciferase, interferon, and Aphthovirus 2A
A single open reading frame polynucleotide encoding a fusion protein comprising a luciferase, an interferon, and an Aphthovirus 2A translational interrupter sequence. The fusion protein can be arranged with luciferase at the N-terminus followed by Aphthovirus 2A and interferon or vice versa, wherein the luciferase may have its N-terminal methionine initiation site removed.
Polynucleotide encoding a fusion protein with secretable luciferase replacing the native secretion sequence
A polynucleotide encoding a fusion protein where the secretable luciferase is fused directly at the N-terminus to a modified polypeptide of interest that has its native N-terminal secretion peptide sequence removed. The removed secretion sequence is replaced by the luciferase secretion sequence with direct adjacency of luciferase and modified polypeptide coding sequences.
Fusion protein comprising secretable luciferase fused to modified polypeptide of interest
A fusion protein comprising, from N- to C-terminus, a secretable luciferase directly fused to a modified polypeptide of interest lacking its native N-terminal secretion peptide, wherein the luciferase replaces this secretion peptide. The fusion protein maintains secretion capability and biological activity.
Method for producing secretable fusion proteins in host cells
A method comprising expression of a fusion protein encoding polynucleotide in a host cell, culturing the host cell to express and secrete the fusion protein comprising luciferase fused to a modified polypeptide of interest, and recovering the secreted fusion protein from culture medium.
The claims encompass polynucleotides, fusion proteins, and methods for producing and quantifying fusion proteins composed of luciferase sequences fused directly or linked via translational interrupter sequences to interferons or other polypeptides of interest with secretion domains replaced by luciferases. This enables secretion, luminescent quantification, and biological activity retention of the fusion proteins.
Stated Advantages
Provides easy and accurate determination of absolute concentrations of interferons and biologically active molecules via luminescent output.
Avoids drawbacks of antibody-based assays, including antibody binding variability, cost, and limited shelf life.
Supports secretion and biological activity retention of fusion proteins despite modification.
Enables direct or indirect quantification using luciferase as a correlate or direct reporter of polypeptide concentration.
Reduces sample standardization issues and improves reliability over conventional antibody-based methods.
Documented Applications
Biotherapeutic applications including treatment of diseases such as Foot-and-Mouth Disease, malignant melanoma, hepatitis B and C, and viral infections in domestic and wild animals.
Diagnostic and quality control applications in molecular quantification of interferons and proteins of interest.
Use in veterinary medicine for treatment of infections such as Feline Herpesvirus 1, Feline infectious peritonitis, Feline Immunodeficiency Virus, Feline Leukemia Virus, and Canine papilloma virus.
Methods for evaluating protein expression and concentration in expression systems via luciferase luminescence.
Interested in licensing this patent?