Recombinant expression platform, constructs and methods for expression of difficult to express proteins (DTE-PS)
Inventors
Arora, Kajal • Kundu, Prabuddha Kumar • RASTOGI, Ruchir • Arora, Nupur Mehrotra
Assignees
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Publication Number
US-12559761-B2
Publication Date
2026-02-24
Expiration Date
Abstract
The present invention relates to expression of SARS-CoV like virus proteins [S, M and E] proteins; recombinant polynucleotides, polypeptides; constructs, virus-like particles (VLPs); immunogenic compositions or vaccines comprising Virus Like Particles (VLPs). Method of producing the VLPs/expressing the multi-subunit virus like proteins and method for co-expression of multi-subunit and virus like proteins (VLPs) are also provided. The present invention also provides strategies, methods, systems, kits and combinations for scalable expression, purification and enhanced production of the virus like proteins of SARS-CoV while maintaining their size range and composition. Such multi-subunit VLPs can be utilized to make immunogenic compositions or vaccines.
Core Innovation
The invention provides a versatile recombinant expression platform that uses an engineered protease deficient yeast host cell and an array of one or more episomal or integrated yeast based expression vectors. The engineered host cell has disrupted endogenous genes encoding protease PRB1 and protease PEP4, and includes auxotrophic markers uracil, lysine, adenine and leucine.
The expression vectors are operably linked with one or more promoters selected from Gal1 promoter, ADH2 promoter and Gal10 promoter, where the promoters can be used singly or in combination. The vector comprises a nucleic acid sequence having at least 90% sequence identity to at least one SEQ ID NO selected from SEQ ID NOs 7, 8, 9, 10, 11 and 12, and the nucleic acid sequence directs insertion of full length or truncated polynucleotide sequence into the yeast host cell for expression of at least one target protein.
The platform is configured to allow enhanced expression of difficult to express target proteins of diverse origin and families compared to a control yeast host cell expression. The described platform supports expression across multiple categories of difficult-to-express targets, including membrane bound and surface localized proteins.
Claims Coverage
The independent claim is clm-00001. Its inventive content comprises two main components—an engineered protease deficient yeast host cell with specific protease gene disruptions and auxotrophic markers, and an array of episomal or integrated yeast expression vectors using Gal1, ADH2, and/or Gal10 promoters with nucleic acid sequences meeting a stated sequence-identity threshold to specified SEQ ID NOs, for enhanced expression of difficult-to-express target proteins versus a control yeast host cell.
Engineered protease deficient yeast host with protease gene disruptions and auxotrophic markers
An engineered protease deficient yeast host cell with disrupted endogenous genes encoding protease PRB1 and protease PEP4 and auxotrophic markers uracil, lysine, adenine and leucine.
Episomal or integrated yeast expression vectors with Gal promoters and SEQ ID–identity nucleic acids
One or more episomal or integrated yeast based expression vectors operably linked with one or more promoters selected from Gal1 promoter, ADH2 promoter or Gal10 promoter, wherein the promoters can be used singly or in combination; the vector comprises a nucleic acid sequence having at least 90% sequence identity to at least one SEQ ID NO selected from SEQ ID NOs 7, 8, 9, 10, 11 and 12, which directs insertion of full length or truncated polynucleotide sequence into said yeast host cell for the expression of at least one target protein.
Enhanced expression of difficult to express target proteins versus a control yeast host cell
The platform allows for enhanced expression of difficult to express target proteins of diverse origin and families compared to a control yeast host cell expression.
Protease-deficient Saccharomyces cerevisiae host with PRB1/PEP4 disruptions and auxotrophic markers
The engineered protease deficient yeast host cell is a Saccharomyces cerevisiae yeast host cell with endogenous genes PRB1 and PEP4 disrupted and auxotrophic markers uracil, lysine, adenine and leucine.
Specific difficult to express target: human GPI anchor protein CD59
The difficult to express target protein is the human GPI anchor protein CD59.
Episomal vector configuration using Ura3 selection marker and CYCT1 terminator elements
The episomal expression vector contains a Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori, and a Gal1 promoter.
Target-polynucleotide SEQ ID–linked expression for defined difficult-to-express proteins
One or more target proteins are encoded by nucleic acid sequences selected from SEQ ID NO: 1 (viral structural capsid protein VP7), SEQ ID NO: 2 (membrane bound Neuraminidase), SEQ ID NO: 3 (sodium ion channel receptor Nav1.7), SEQ ID NO: 4 (fatty acid desaturase), SEQ ID NO: 5 (fatty acid elongase), and SEQ ID NO: 6 (GPI anchor protein CD59), for expression in the engineered protease deficient yeast host using episomal or integrated yeast based expression vectors.
Across the claim set provided, the core coverage centers on the combination of an engineered protease deficient yeast host with PRB1/PEP4 disruptions and auxotrophic markers, together with episomal or integrated yeast expression vectors using Gal1/ADH2/Gal10 promoters and nucleic acids defined by at-least-90% identity to SEQ ID NOs 7–12, to achieve enhanced expression of difficult to express proteins versus a control yeast host; dependent claims further narrow host species, example target proteins such as CD59, defined vector/selection components, and an assessment step for surface localization by confocal microscopy.
Stated Advantages
Enhanced expression of difficult to express target proteins of diverse origin and families compared to a control yeast host cell expression.
Documented Applications
Expression and scalable production of SARS-CoV-like structural proteins S, M, and E using the yeast-based recombinant expression platform.
Expression of membrane-anchored influenza neuraminidase (NA), including immunogenicity and enzymatic activity assessment.
Expression and purification of viral capsid glycoprotein VP7 with yield scaling.
Expression of the transmembrane ion channel receptor Nav1.7 with localization to the cell surface (plasma membrane localization) assessed by confocal microscopy.
Expression of fatty acid desaturase and fatty acid elongase, including scale-up and purification.
Expression of a GPI-anchored protein CD59.
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