Baculovirus system for expressing proteins forming virus-like particles
Inventors
Gomez Sebastian, Silvia • López Vidal, Javier • Martinez Escribano, José Angel
Assignees
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Publication Number
US-9879280-B2
Publication Date
2018-01-30
Expiration Date
Abstract
The present invention may be included in the field of biotechnology and it covers the improved production of recombinant proteins in insect cells or insect larvae as biofactories by a novel expression cassette. This expression cassette comprises nucleic acid sequences such as promoters, homologous regions (hr) as enhancers, and sequences encoding transcriptional regulators, for example, the baculovirus Ac-ie-01 cDNA, or any combination thereof, which are able to increase the quality and production efficiency of the recombinant proteins. Moreover, the present invention is also directed to the vectors themselves comprising the above mentioned nucleic acid sequences of the invention, cells or insects infected, transformed or transfected with those sequences or vectors, and methods for producing the recombinant proteins by using the aforesaid sequences, vectors, cells or insects.
Core Innovation
The invention relates to an improved baculovirus expression vector system (BEVS) expression cassette. The cassette comprises a first nucleic acid sequence encoding baculovirus transcriptional regulators derived from Ac-ie-01 and/or related IE-1 and IE-0 regulators, and a second nucleic acid sequence directing expression of a recombinant virus-like particle (VLP) protein. The regulators are described as being overexpressed above endogenous levels to improve recombinant protein quality and productivity.
The expression cassette further comprises at least one recombinant homologous region (hr) as an enhancer region operably linked to a promoter suitable for driving recombinant protein expression. The promoter is described as selected from promoters that include p10 and chimeric p10-containing promoters, in combination with hr1 to increase output, including recombinant VLP protein yields and quality. The disclosed BEVS component sets are supported by sequence elements and enhancer homologous region selections referenced by identity thresholds.
The described system is reported to increase host cell proliferation and viability and to reduce cytopathic effect/cell death following infection with recombinant baculoviruses carrying the IE-1/IE-0 overexpression cassette. Documented comparisons are described as showing substantially higher expression/productivity of VLP-related capsid proteins than conventional BEVS approaches using other promoters such as polh. The partial content also describes recovery/biomass increases in insect larvae and quantified improvements in yields and survival/productivity in trichoplusia ni (T. ni) larvae.
Claims Coverage
Independent claim coverage includes three independent expression-cassette claim positions, each defining a baculovirus expression vector system (BEVS) cassette with a defined first nucleic acid sequence operably linked to a second nucleic acid sequence encoding a virus-like particle (VLP) protein, and one claim further requiring an enhancer homologous region and a promoter selected by a defined sequence reference.
Engineered expression cassette for VLP production in BEVS
An expression cassette for a BEVS comprising a first nucleic acid sequence selected from SEQ ID NO:1/95% identity variants or SEQ ID NO:6/95% identity variants, and a second nucleic acid sequence that directs expression of a recombinant virus-like particle protein, wherein the expression cassette also comprises at least one recombinant homologous region (hr) as an enhancer region operably linked to a promoter suitable for driving the expression of the recombinant protein, and wherein the promoter is selected from SEQ ID NO:13 or a promoter sequence having at least 95% sequence identity to SEQ ID NO:13.
Functional-retention IE regulator sequence linked to VLP coding sequence
An expression cassette for a BEVS comprising a first nucleic acid sequence selected from SEQ ID NO:19 or a nucleic acid sequence substantially retaining the activity of the functional elements and having at least 95% sequence identity to SEQ ID NO:19, and a second nucleic acid sequence that encodes a recombinant virus-like particle, wherein the first nucleic acid sequence is operably linked to the expression of the second nucleic acid sequence.
High-identity functional element nucleic acid sequence in a BEVS cassette
An expression cassette for a BEVS comprising a nucleic acid sequence selected from SEQ ID NO:51 or a nucleic acid sequence substantially retaining the activity of the functional elements and having at least 95% sequence identity to the sequence of SEQ ID NO:51.
Across the independent claims, the core BEVS claim coverage centers on specific first nucleic acid sequences with sequence identity or activity constraints and operable linkage to a second nucleic acid sequence encoding a virus-like particle. One independent claim additionally requires an enhancer homologous region operably linked to a promoter selected from a defined SEQ ID NO:13 set.
Stated Advantages
Increases host cell proliferation and viability.
Reduces cytopathic effect/cell death.
Increases recombinant protein recovery/biomass in insect larvae.
Increases expression/productivity of VLP-related capsid proteins versus conventional BEVS using other promoters.
Improves survival/productivity in T. ni larvae.
Documented Applications
Production of recombinant virus-like particle (VLP) proteins in a baculovirus expression vector system for use in insect larvae, including T. ni, with recovery/biomass and survival/productivity improvements reported.
Production/expression of specific VLP-related proteins identified in the partial content, including PCV2 Cap (ORF2/Cap), RHDV VP60, and HPV16 L1.
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