Baculoviral DNA elements for the expression of recombinant proteins in a host cell
Inventors
Gomez Sebastian, Silvia • López Vidal, Javier • Martinez Escribano, José Angel
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9982239-B2
Publication Date
2018-05-29
Expiration Date
Abstract
Reagents and methods are provided that allow for an improved expression of a recombinant protein. More specifically, the introduction of recombinant DNA elements into a host cell allows for the increased expression of a recombinant protein, an improvement of the correct folding of said protein and an increase in cell viability and proliferation of the host cell, These recombinant DNA elements can be introduced into host cells, for example, via a recombinant baculovirus, which has incorporated said elements. The recombinant DNA elements include nucleic acids encoding transcriptional regulators, such as IE-0 and IE-1, transcriptional enhancer elements, such as the homologous region (hr) and promoters.
Core Innovation
The invention relates to a recombinant baculovirus that includes one native copy of an Ac-ie-01 gene in its genome and an additional copy of the Ac-ie-01 gene under control of a promoter. The recombinant baculovirus is capable of expressing increased levels of immediate early protein (IE-1) or immediate early protein 0 (IE-0) in a host cell infected with the recombinant baculovirus compared to a host cell infected with a control baculovirus.
The extra Ac-ie-01 nucleic acid is selected from nucleic acid sequences comprising SEQ ID NOS: 2-5 and/or nucleic acid sequences encoding an amino acid sequence comprising SEQ ID NOS: 6-9. The first nucleic acid sequence further comprises at least one recombinant homologous region (hr) from a baculovirus used as an enhancer region, with the recombinant homologous region (hr) operably linked to a promoter suitable for driving the expression of a recombinant protein.
Enhanced immediate early protein expression is achieved in insect host cells derived from Trichoplusia ni or Spodoptera frugiperda. The disclosed embodiments describe improved recombinant protein expression that shifts expression earlier (early-to-late), improved cell proliferation/viability while preserving cell integrity, and improved post-translational processing and folding of a recombinant protein.
Claims Coverage
The independent claim covers one inventive feature set centered on a recombinant baculovirus engineered to increase immediate early protein expression by adding an extra Ac-ie-01 gene sequence under promoter control, including an enhancer homologous region operably linked to the promoter, and limited to specified insect host cells and sequence selections.
Extra Ac-ie-01 copy for increased immediate early protein expression
A recombinant baculovirus comprising one native copy of an Ac-ie-01 gene in its genome and a first nucleic acid sequence having an extra copy of the Ac-ie-01 gene under control of a promoter, wherein infected host cells express increased levels of immediate early protein (IE-1) or immediate early protein 0 (IE-0) compared to infection with a control baculovirus containing only one native Ac-ie-01 gene copy and lacking the first nucleic acid sequence.
Defined Ac-ie-01 sequence variants and enhancer homologous region linked to a promoter
The first nucleic acid sequence comprises a nucleic acid sequence selected from nucleic acid sequences comprising SEQ ID NOS: 2-5 or nucleic acid sequences encoding an amino acid sequence comprising SEQ ID NOS: 6-9, and further comprises at least one recombinant homologous region (hr) from a baculovirus as an enhancer region operably linked to a promoter suitable for driving recombinant protein expression.
Promoter restricted to SEQ ID NOS: 10-16 with specified insect host species
The promoter is a nucleic acid sequence comprising any of SEQ ID NOS: 10-16, and the host cell is derived from an insect belonging to Trichoplusia ni or Spodoptera frugiperda.
The core coverage is a recombinant baculovirus that augments immediate early protein expression by adding an extra Ac-ie-01 copy under a defined promoter, with an hr enhancer region operably linked to that promoter, in host cells derived from Trichoplusia ni or Spodoptera frugiperda.
Stated Advantages
Increased levels of immediate early protein (IE-1) or immediate early protein 0 (IE-0) in host cells infected with the recombinant baculovirus compared to a control baculovirus.
Improved recombinant protein expression that shifts expression earlier (early-to-late).
Increased cell proliferation/viability at late post-infection while preserving cell integrity.
Improved post-translational processing and folding of a recombinant protein, indicated by improved GFP band integrity/functionality.
Documented Applications
No documented applications found
Interested in licensing this patent?