Expression of recombinant proteins in Trichoplusia ni pupae
Inventors
Martínez Escribano, José Ángel • ALVARADO FRADUA, Carmen • REYTOR SAAVEDRA, Edel • CID FERNANDEZ, Miguel
Assignees
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Publication Number
US-11278014-B2
Publication Date
2022-03-22
Expiration Date
Abstract
The present invention covers means and methods to increase the efficiency of recombinant protein expression, in particular to optimize the industrial production of recombinant proteins in insect pupae, particularly in Trichoplusia ni (T. ni) pupae. Moreover, the present invention is also directed to the pupae itself comprising baculovirus, pupae infected, transformed, transduced or transfected with baculoviruses or bacmids, as well as devices suitable for performing the methods of the present invention.
Core Innovation
The invention provides an insect-pupa recombinant protein production platform using silk-free pupae of genus Trichoplusia that are infected or injected with AcMNPV-derived recombinant baculoviruses or bacmids. The silk-free pupa is produced by removal of cocoon silk, and the recombinant baculovirus and/or bacmid is injected into the silk-free pupa.
The core construct boost is achieved by including nucleic acid elements that overexpress baculovirus transcriptional regulators IE-1 and IE-0 and/or fragments above endogenous levels during baculovirus infection. The disclosure further provides a recombinant homologous region (hr1) operably linked to a promoter suitable for driving expression of a recombinant protein, with promoter and cassette compositions defined using sequence identities and/or SEQ ID NOs.
The disclosure also ties the approach to downstream harvesting and purification after expression, and to pupae-derived baculovirus inoculum without insect cell culture. Examples show improved expression yields for recombinant proteins including GFP, Cap, HA, and VLP production such as VP60.
Claims Coverage
The provided independent claims cover three inventive themes: silk-free Trichoplusia pupae containing injected AcMNPV-derived recombinant baculovirus and/or bacmid with specified overexpression and hr-promoter features; methods for producing recombinant proteins by injecting silk-free Trichoplusia pupae with recombinant AcMNPV baculovirus and incubating to express recombinant proteins; and a method for producing a silk-free pupa comprising recombinant baculovirus by injecting an AcMNPV-derived bacmid and incubating to produce recombinant baculovirus. Across these claims, the inventive features include IE-1/IE-0 overexpression above endogenous levels, a recombinant homologous region (hr1) operably linked to a suitable promoter for recombinant protein expression, and sequence-identity and SEQ ID NO limitations on the nucleic acid elements.
Injected silk-free pupa of genus Trichoplusia with AcMNPV-derived recombinant baculovirus and/or bacmid
A silk-free pupa comprising a recombinant baculovirus and/or a bacmid derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), wherein the silk-free pupa belongs to genus Trichoplusia, and wherein the recombinant baculovirus and/or bacmid was injected into the silk-free pupa.
IE-1/IE-0 overexpression above endogenous level with hr operably linked to a promoter
A silk-free pupa wherein the recombinant baculovirus and/or a bacmid comprises a nucleic acid sequence that allows for expression, above an endogenous level, of IE-1, IE-0, and/or fragments thereof, and a recombinant homologous region (hr) operably linked to any promoter that is suitable for driving expression of a recombinant protein.
Sequence-defined IE-1/IE-0 nucleic acid elements and hr1-promoter driving recombinant protein expression
The silk-free pupa wherein the nucleic acid sequence comprises SEQ ID NOs: 1-5 or nucleic acid sequences having at least 80% sequence identity with SEQ ID NOs: 1-5 and encoding a protein able to function as a transcriptional regulator in a recombinant baculovirus, and wherein the nucleic acid sequence encoding an amino acid comprises SEQ ID NOs: 6-9 or amino acid sequences having at least 70% sequence identity with SEQ ID NOs: 6-9 and being able to function as a transcriptional regulator in a recombinant baculovirus.
Producing at least one recombinant protein by injecting silk-free Trichoplusia pupae with recombinant AcMNPV baculovirus and incubating for expression
A method for producing at least one recombinant protein, comprising providing a silk-free pupa of genus Trichoplusia; injecting the silk-free pupa with a recombinant baculovirus derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to obtain an inoculated silk-free pupa, wherein the recombinant baculovirus comprises a nucleic acid sequence encoding at least one recombinant protein; and incubating the inoculated silk-free pupa for a period of time sufficient for the at least one recombinant protein to be expressed.
Producing a silk-free pupa comprising recombinant baculovirus by injecting an AcMNPV-derived bacmid and incubating
A method for producing a silk-free pupa comprising a recombinant baculovirus, comprising providing a silk-free pupa belonging to genus Trichoplusia; injecting the silk-free pupa with a bacmid suitable for producing a recombinant baculovirus derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to obtain an inoculated silk-free pupa; and incubating the inoculated silk-free pupa for a period of time sufficient to produce the recombinant baculovirus.
Across the independent claims, the inventive coverage centers on silk-free Trichoplusia pupae that are injected with AcMNPV-derived recombinant baculovirus and/or bacmid, with recombinant baculovirus elements enabling overexpression of IE-1 and IE-0 and/or fragments above endogenous levels as transcriptional regulators, together with a recombinant homologous region (hr) operably linked to a promoter suitable for driving recombinant protein expression. Additional restrictions specify SEQ ID NO-defined nucleotide/protein sequences or minimum sequence identity thresholds, and the broader method claims cover producing at least one recombinant protein and producing recombinant baculovirus within the silk-free pupa by injection and incubation.
Stated Advantages
Documented Applications
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