WO1995021249A1 - Cassette pour accumuler de multiples proteines par synthese d'un polypeptide a traitement auto-proteolytique - Google Patents

Cassette pour accumuler de multiples proteines par synthese d'un polypeptide a traitement auto-proteolytique Download PDF

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WO1995021249A1
WO1995021249A1 PCT/US1995/001495 US9501495W WO9521249A1 WO 1995021249 A1 WO1995021249 A1 WO 1995021249A1 US 9501495 W US9501495 W US 9501495W WO 9521249 A1 WO9521249 A1 WO 9521249A1
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vector
sequence
cell
nia
nucleotide sequence
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Roger N. Beachy
Jose F. Marcos
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/34011—Potyviridae
    • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to methods for plant transformation to enhance and control gene expression. More particularly, this invention relates to a method for expressing more than one transgenic gene in plants in equimolar amounts from a single promoter.
  • a cassette expression vector based on the nuclear inclusion (NIa) protease from tobacco etch virus (TEV) allows the transcription and translation of a nucleotide sequence comprising the TEV NIa coding region flanked on each side by its heptapeptide cleavage sequences and insertion sites for in frame insertion of two different open reading frames coding for heterologous proteins.
  • TEV tobacco etch virus
  • the protease releases the two heterologous proteins in equimolar amounts by autoproteolytic reaction. Therefore, the invention provides a method for obtaining equimolar amounts of different proteins expressed under the control of a common promoter.
  • a plurality of insertions sites can be engineered into a cassette containing a single TEV
  • the expression cassette functions to express genes encoding two or more different heterogeneous peptides from a single polypeptide by post translational self-cleavage by the NIa protease.
  • Figure 1 A is a schematic diagram of a TEV-NIa-based expression cassette vector pPROl .
  • the open box represents the NIa open reading frame.
  • the shaded areas enlarged above show (as both nucleotide and amino acid sequence) the heptapeptide recognition sequence for the NIa proteolytic activity at both N- and C-termini of NIa; the engineered Sma I and
  • Stu /cloning sites (underlined) for the in frame introduction of different genes; and start ATG and stop TGA codons.
  • the NIa processing site between Gin and Gly is indicated as an open arrowhead.
  • the sequence of the TEV 5' non-translated region is also indicated by a black arrow upstream of the NIa coding sequence. Relevant unique restriction enzyme sites are indicated: Ba (BamHl), Bg (Bgl II), Ec (EcoR I), Sa (Sal I), Sc (Sac I),
  • Figure IB is a detailed restriction map of pPROl displaying the nucleotide sequence and the amino acid sequence of the NIa protease (SEQUENCE I.D. NO. 6).
  • Figure IC is a schematic diagram showing amino acid additions that result at N- and C- termini of proteins cloned at the Sma I or Stu I enzyme restriction insertion sites of expression vector pPROl upon translation and subsequent proteolytic processing.
  • the amino acid represented by X depends upon the particular restriction site used for cloning and can be coincident with amino acids in the cloned proteins in some cases.
  • Figure 2 shows an autoradiograph of an SDS-PAGE gel indicating the results of in vitro translation of RNA transcribed from the pPROl expression cassette.
  • Translation reactions were programmed with 1 ⁇ g of brome mosaic virus (BMV) RNAs (lane B), with no RNA added (lane 0), and with RNA transcribed in vitro from pPROl (lane 1).
  • BMV brome mosaic virus
  • RNAs and the position of the 49 kDa TEV NIa protein are indicated.
  • Figure 3A shows a schematic representation of six different polypeptides translated transcribed in vitro from different pPROl -derived constructs containing the TMV CP sequence.
  • Open boxes represent the TEV-NIa sequence.
  • Striped boxes represent the TMV CP sequence contained in the insertion site.
  • the names of the constructs and the expected molecular mass of the translated and processed products are indicated.
  • Q/G indicates the amino acid residues at the cleavage sequence in constructs cloned in pPROl ; whereas H/G indicates the His to Gin mutation at -1 position that inhibits processing by NIa in constructs cloned in pPRO4.
  • Figure 3B shows an autoradiograph of an SDS-PAGE gel containing in vitro translation products obtained from the constructs shown in Figure 3 A.
  • the vertical axis and lane assignments are the same as described for Figure 3C below.
  • Figure 3C shows fluorographs of immunoprecipitation analyses using anti-TMV CP antibody with aliquots from the translation samples shown in Figure 3B.
  • translation reactions were programmed with no RNA added (lane 0); with RNA transcribed in vitro from pPROl (lane 1); pPROl.NT (lane 2); pPROl.TN (lane 3); pPRO 1.T ⁇ N (lane 4); pPRO4.NT (lane 5); and pPRO4.TN (lane 6).
  • T TMV coat protein
  • N NIa protease
  • Figure 4 shows the results of in vitro translation of RNAs transcribed from pPROl constructs containing TMV CP and SMV CP coding sequences inserted at two sites in the cassette.
  • Figure 4 A is a schematic diagram representing the vectors pPROl .SNT and pPROl .TNS.
  • the open box represents the TEV-NIa sequence.
  • Striped and dotted boxes represent TMV CP and SMV CP sequences, respectively that have been inserted into the cassette insertion sites.
  • S SM coat protein.
  • Figure 4B shows an autoradiograph of an SDS-PAGE gel with in vitro translation products obtained from pPRO 1. SNT and pPRO 1.TNS vectors. Translation reactions were programmed with no RNA added (lane 0); with RNA transcribed in vitro from pPROl (lane 1); pPROl.SNT (lane 2); and pPROl.TNS (lane 3). The molecular mass (in kDa), positions of the major proteins translated from BMV RNAs, and the positions of the TEV NIa, SMV CP and TMV CP are indicated.
  • Figure 5 shows the results of in vitro translation of RNAs transcribed from a pPROl vector containing SMV CP and uic A. ( ⁇ -glucuronidase, GUS) coding sequences in the two insertion sites.
  • Figure 5B shows an autoradiograph of an SDS-PAGE gel with in vitro translation products obtained from cassette vector pPROl .SNG. Positions of TEV NIa, GUS, and SMV CP proteins are indicated. Translation reactions were programmed with no RNA added (lane 0); and with RNA transcribed in vitro either from pPROl (lane 1); or pPROl.SNG (lane 2). Molecular mass (indicated in kDa), and positions of proteins translated from BMV RNAs is indicated: TEV NIa, GUS, and SMV CP proteins are also indicated. A black arrowhead indicates the position of a 110 kDa polypeptide present in small amounts.
  • Figure 5C shows a photograph of an SDS PAGE gel used in a time course in vitro translation reaction with vector pPROl.SNG. Samples were withdrawn at times (in minutes) indicated at the top of each lane. At an incubation time of 15 minutes on SDS-
  • TEV the NIa protease is synthesized as part of the polyprotein that results from the translation of the TEV genome.
  • the genomic sequence of TEV first disclosed by R. Allison, et al. (Virology, 154:9-20, 1986) is publicly available from EMBL and Genebank database under accession number Ml 5239.
  • NIa recognizes and cleaves specific sequences of seven amino acids (heptapeptide) contained in the polyprotein and is responsible for partial processing of the viral polyprotein.
  • Heptapeptide cleavage sequences recognized by the NIa from TEV have been shown to be Glu-X-X-Tyr-X-Gln-Gly (SEQUENCE I.D. NO. 1) or Glu-X-X-Tyr-X- Gln-Ser (SEQUENCE I.D. NO.2) wherein X can be any amino acid (J. Carrington, et al,
  • Cleavage location by TEV-NIa protease is after the Glu amino acid.
  • the self- recognized cleavage sequence at the N-terminini of the NIa protease is Glu-Pro-Val-Tyr- Phe-Gln-Gly (SEQUENCE I.D. NO. 3) and the self-recognized cleavage sequence at the C-termini is Glu-Leu-Val-Tyr-Ser-Gln-Gly (SEQUENCE I.D. NO. 4).
  • These two heptapeptides are the ones that bracket the NIa protein in the TEV polyprotein.
  • NIa releases itself from the polyprotein in an autoproteolytic reaction attacking at the cleavage sequences (J. Carrington, et al, Virology, 16Q:355-362, 1987), and is active both in cis, processing polypeptides in which it is included, and in trans, simultaneously cleaving different polypeptides.
  • the cis protease activity of NIa has been assayed with different TEV polyproteins produced in vitro which contained NIa and either naturally occurring or mutated versions of the cleavage sequence (J. Carrington, et al, J. Virology, 1988, 1987, supra).
  • TEV-NIa based expression cassette provided herein has been constructed to exploit the protease activity of NIa in a self-processing polypeptide in order to express two or more different proteins in equimolar amounts.
  • cassette vector named pPROl, shown in Figure 1
  • pPROl was obtained by PCR amplification using as template a full length TEV cloned cDNA. It comprises PRO1 (SEQUENCE ID NO. 5), which includes an open reading frame encompassing the NIa sequence (TEV nucleotides 5673 to 6983 as numbered in R.
  • the TEV-NIa based cassette described herein also provides at least two blunt end restriction sites, preferably unique, that allow the in frame insertion of heterologous protein sequences vector for expression as part of a self-processing polypeptide.
  • heterologous shall have the meaning that the gene inserted into the cassette insertion site is not native to TEV.
  • one insertion site is provided by a Sma I restriction enzyme site at the N-terminus of the TEV NIa sequence, and the other insertion site is provided by a
  • the cassette optionally provides a start codon, preferably ATG, and a stop codon, preferably TGA, engineered upstream of the 5-prime site and downstream of the 3-prime site, respectively.
  • a start codon preferably ATG
  • a stop codon preferably TGA
  • an ATG start codon is upstream of the Sma I site
  • a TGA stop codon is downstream of the Stu I site.
  • the TEV-NIa based vectors herein preferably include upstream of the open reading frame the 144 nucleotide 5' non-translated region from TEV RNA, which has been shown to enhance translation in vitro and in vivo (J. Carrington and D. Freed, J. Virol, 64:1590-1597, 1990).
  • NIa protease In the embodiment of the invention utilizing more than one restriction site on one or both sides of the gene encoding the NIa protease and its flanking self-recognition sequences, it will be necessary to provide additional NIa protease self-recognition sequences between adjacent recognition sequences to allow for post translational self-cleavage by the NIa protease.
  • a single protease is sufficient to cleave multiple sites within the single polypeptide produced from expression of the cassette.
  • PRO1 ( Figure IB; SEQUENCE ID NO. 6) was sequenced using techniques known in the art, and six mutations from the native sequence previously published for TEV were found. These changes were, according to numbering in Allison, supra, GC to CG at nucleotide 5768-5769, A to G at nucleotide 5773, A to G at nucleotide 6235, T to C at nucleotide
  • NIa is a highly specific proteinase whose cleavage sequence has been well characterized (Carrington, et al, 1988; Dougherty, et al, 1988, supra; W. Dougherty, et al, Virology, 111:356-364, 1989; Dougherty, et al, Virology, 172:145-155, 1989).
  • NIa retains activity in vitro when cleavage sequences are inserted into several locations in TEV polyproteins (Carrington, et al, 1988, supra; Dougherty, et al, 1988, supra) or into non-viral proteins (Parks, et al, J. Gen. y ' irol., 72.:775-7$3, 1992).
  • Nla cleaves its substrate heptapeptide properly in vivo when expressed as a transgene in plants (Restrepo-Hartwig, et al, J. Virology, 66:5662-5666, 1992).
  • the NIa protease functions in vitro to cleave polypeptides containing inserted coding sequences for many different polypeptides ranging in size from 1 to as many as about 800 amino acids. In most of the constructs tested, cleavage was so effective that non- processed precursors could not be detected. In only two cases (an illustration is shown with pPROl,SNG in Example 4) were minimal amounts of non-cleaved precursors detected, indicating a lack of complete processing. These in vitro results suggest utility of this approach for in vivo applications as well wherein the vectors are introduced into suitable plants by electroporation into plant protoplasts using methods well known in the art.
  • Transformed protoplasts can be harvested and grown into full transgenic plants (C. A. Rhodes, et al, Science 240:204-207, 1988).
  • NIa-based expression cassette vectors are used in systems other than those involving plant cells.
  • the expression cassette of this invention can be used in any system in which the NIa protease has activity, for example, insect bacteria, mammalian, and other eukaryotic cells if operatively linked to suitable expression control elements such as a promoter, and a polyadenylation sequence, so as to bring about replication of the attached segment in a vector suitable for the type of cell line selected.
  • suitable expression control elements such as a promoter, and a polyadenylation sequence
  • vector to which a cassette of this invention is operatively linked depends directly, as is well known in the art, on the host cell to be transformed and the functional properties desired, e.g., vector replication and protein expression, these being limitations inherent in the art of constructing recombinant molecules.
  • the vector itself may be of any suitable type, such as a viral vector (RNA or DNA), naked straight-chain or circular
  • lipid vesicles such as liposomes
  • liposomt ay be targeted to particular cells using other conventional techniques, such as providing an antibody or other specific binding molecule on the exterior of the liposome (see, e.g., A. Huang, et al, J. BioL Chem., 255:8015-8018, 1980).
  • transient expression is contemplated wherein expression of the polypeptide is driven either by conventional transcriptional promoters or by plant viral vectors.
  • the TEV-NIa based cassette vector is used in prokaryotic systems since NIa proteases from different potyvirus have been shown to be active when expressed in bacterial cells (Garcia, et al, Virology, 170:362-369, 1989;
  • the TEV NIa based expression vector can be advantageously used, therefore, whenever it is desirable to achieve equimolar production of two peptides in bacterial expression systems by inse .-ng the NIa cassette into a bacterial expression vector, such as members of the pUC vector family.
  • a bacterial expression vector such as members of the pUC vector family.
  • Other insect and animal cells known in the art to be useful in expression of recombinant proteins can also be used.
  • the cassette vectors can be used in production of recombinant antibodies wherein it is desirable to achieve equimolar amounts of the heavy and light chains.
  • the cassette vectors provided herein are used to produce molecules that spontaneously assemble a two subunit complex, such as an enzyme.
  • a vector having more than two insertion sites is used to express multimers of any type.
  • Proteins expressed in the cassette vectors of this invention contain additional or extraneous amino acid residues at both N- and C-termini as a consequence of the NIa target heptapeptide and the cloning strategy used.
  • the schematic diagram of Figure IC illustrates the amino acid additions at N- and C-termini that result when in the proteins (open boxes) are cloned at either Sma I (Sm) or Stu I (ST) insertion sites of pPRO 1.
  • the amino acid represented by 'X' will depend on the restriction site used for cloning. In some cases one or more of the extraneous amino acids can be incorporated into the protein because it is already native to its sequence and would not have to " be engineered in.
  • the biological activity of some proteins expressed in this system may be affected.
  • one skilled in the art will know how to purify the produced proteins and treat them to clip off the extraneous residues.
  • the heterogenous proteins after cleavage by the protease can have among the extraneous terminal amino acids an undefined amino acid (represented by 'X') immediately next thereto at either end. If 'X' is selected to be a methionine and the produced peptide contains no other methionines, the peptide can readily be treated with cyanogen bromide to remove the extraneous residues.
  • the coat protein of TMV which contains no methionines, can be expressed in one or both of the insertion sites, purified, and then can be treated with cyanogen bromide to provide the coat protein sequence free of extraneous terminal residues.
  • cyanogen bromide One skilled in the art will be able to similarly utilize enzymes that cleave peptides between two particular residues to clip off the terminal extraneous residues from product heterogeneous peptides.
  • coat protein mediated resistance (CPMR) to viral infections can generally be obtained only against viruses of the same taxonomic group as the one whose coat protein was used as the vaccine (Fitchen & Beachy, Annu. Rev. Microbiol, 47:739-763, 1993).
  • CPMR coat protein mediated resistance
  • sequences encoding two or more viral coat proteins from different taxonomic groups can be inserted into insertion sites of a NIa-based vector having two or more insertion sites.
  • an insect resistance gene can be combined with a virus resistance gene.
  • the vector of this invention can be used to express a selectable marker plus any other gene encoding a protein of the size contemplated herein.
  • the vector into which the cassette is ligated is a modification of the "infectious cDNA clone" of the tobacco mosaic virus to which is operably linked the promoter of the T7 polymerase.
  • Ul (common) strain of TMV have been produced in vitro using bacteriophage T7 RNA polymerase (Dawson, et al, Proc. Natl. Acad. Sci USA, 83 : 1832-1836, 1986; Meshi, et al, Proc. Natl. Acad. Sci. USA, 83:5043-5047, 1986).
  • this transcript causes systemic viral infection. Therefore, the vector of this invention can also be used to simultaneously provide systemic resistance to insect and virus in plants when inserted into the infectious cDNA clone of TMV.
  • the cDNA encoding the TMV movement protein is deleted from the TMV infectious clone, and the NIa-based cassette is ligated in its place, thereby creating a modified viral vector.
  • Nucleotide sequences encoding heterologous peptides ligated into the insertion sites of the NIa-based cassette contained within the modified infectious clone can be inoculated into host plants for expression therein.
  • the coat proteins of plant viruses belonging to a different taxonomic group than TMV, or other genes capable of protecting a plant against insect or disease can be ligated into the insertion sites of the NIa-based cassette in the infectious clone vector for production in the host plant. Since the modified infectious clone vector retains the native gene encoding the coat protein of TMV, a cassette with two insertion sites can be used to express multiple CP sequences confer CPMR against viruses from three different taxonomic groups.
  • recombinant plants transformed with a gene encoding the wild type movement protein of the TMV such as plant line 277 (Deom, et al, Cell, 69:221-224, 1992) are inoculated with the modified infectious clone vector, the viral infection will spread systemically.
  • This modified infectious clone vector takes advantage of the extremely high level of expression characteristic of the viral system, and can be used to economically produce large amounts of polypeptides, virions suitable for use as vaccines, etc.
  • product polypeptides and/or virions can be purified from plant leaves using standard methods (Bruening, et al, Virology, 71:498-517, 1976).
  • constructs containing NIa and the CP of TMV were introduced in Nicotiana tabacum via Agrobacterium tumefaciens transformation.
  • Preliminary data indicate that TMV CP expressed in vivo as part of pPROl confers CPMR (data not shown).
  • Additional constructs with an insert that encodes a viral coat protein and a gene encoding ⁇ -glucuronidase will enable use of GUS activity as a probe for the levels of expression of the CP. Since the activity of the CP is destroyed if the protease does not cleave in the exact place anticipated, this experiment showed the specificty of the NIa protease for cleaving multiple exogenous peptides.
  • the expression cassette vector pPROl ( Figures 1 A and IB) was assembled in pBluescript II KS (+) (Stratagene, San Diego, CA) under the transcriptional control of a T7 promoter by directional insertion of PRO 1 (SEQUENCE ID NO. 5) at the Sac I - EcoR I sites of the multiple cloning site, rendering pPROl .
  • NIa and 5'-non-translated (5-NTR) sequences from TEV were obtained by PCR using as DNA template a full length TEV cDNA clone
  • Oligonucleotide primers for amplification of NIa were TEVNIA.N and TEVNIA.C (SEQUENCE ID NOS. 7 and
  • TEVNIA.N Stu I and EcoR I (TEVNIA.C) restriction enzyme sites.
  • the PCR product was directionally inserted pBluescript using Xba I and EcoR I to yield vector pBCNIa.
  • Ohgonucleotide primers used for PCR amplification of the 5'-NTR of TEV were TEVNTR.5 and TEVNTR.3 (SEQUENCE ID NOS. 9 and 10, respectively). These primers contained either Sac I and Bgl II (TEVNTR.5) or Sma I (TEVNTR.3) restriction enzyme cleavage sites.
  • the final step in the assembly of pPROl was a Sac 1-Sma I directed insertion of the TEN-5 ⁇ TR resulting from the PCR reaction into vector pBC ⁇ Ia. Mutagenesis at the heptapeptides in the TEV sequence encoding the protease cleavage recognition sites was accomplished with primers TEV ⁇ IA. ⁇ 2 and TEVNIA.C3 (SEQUENCE ID NOS.
  • the cDNAs for different open reading frames (ORFs) encoding heterogenous peptides inserted into pPROl included those encoding tobacco mosaic virus (TMV) and soybean mosaic virus (SMV) coat proteins (CP), as well as the uidA gene encoding the ⁇ - glucuronidase (GUS) activity from E. coli.
  • ORFs open reading frames
  • These ORFs were obtained by PCR using as template publicly available nucleotide sequences.
  • the nucleotide sequence of tobacco mosaic virus RNA first published by P. Goelet, et al. (Proc. Natl. Acad. Sci.
  • primers TMV CP 51 SEQUENCE ID NO. 13 was used at the 5' end and TMV CP 31 (SEQUENCE ID NO. 14) was used at the 3* end.
  • primer SMV CP Nl SEQUENCE ID NO. 15 was used at the 56* end and primer SMV CP C2 (SEQUENCE ID NO. 16) was used at the 3' end.
  • primer GUS N2 SEQUENCE ID NO. 18 was used at the 5' end and primer GUS Cl (SEQUENCE ID NO. 19) was used at the 3' end.
  • TEVNIA.C 5'-GCGAATTCAAGGCCT CCCTTGCGAGTACACCAATTCA-3' (SEQ. ID NO. 8)
  • TEVNTR.3 5'-TCCCCCGGG CATGGCTATCGTTCGTAAATGG-3' (SEQ. ID NO. 10)
  • TEVNIA.N2b 5'-TGGCCCGGG GAACCAGTCTATTTCCATGGG-3' (SEQ. ID NO. 11 )
  • TEVNIA.C3 b 5'-GCGAATTCAAGGCCT CCCATGGGAGTACACCAATTCA-3 (SEQ. ID NO. 12)
  • TMVCP.31 5'-AGGCCCGGG AGTTGCAGGACCAGAGGTCC-3' (SEQ. ID NO. 14)
  • SMVCP.N 1 5'-AAAGGCCT TCAGGCAAGGAGAAGG-3' (SEQ. ID NO. 15)
  • GUS.C1 5'-CGGAATTC TCATTGTTTGCCTCCCTGCTG-3' (SEQ. ED NO. 18)
  • nucleotides annealing to the target genes are underlined with a single line, whereas nucleotides corresponding to the restriction enzyme recognition sequences are doubly underlined.
  • TEVNIA.N. and TEVNIA.C are marked by an asterisk underneath.
  • PCR products corresponding to SMV- and TMN-CP genes were digested with Stu I and Sma I and inserted either at the Sma I or the Stu I sites of pPROl ( Figure 1), depending on the construct.
  • the PCR product corresponding to the uidA ORF was digested with Stu I and EcoR I and inserted at the C terminus of ⁇ la in pPROl .
  • plasmid pPROl DNA containing the inserted heterologous ORFs purified from E. coli through QIAprep mini columns was first linearized with Sal I (which cleaves downstream of pPROl), and subsequently transcribed in vitro with T7 RNA polymerase (Epicentre Technologies, Madison, WI). Size and integrity of transcribed mRNA were confirmed by agarose gel electrophoresis. Approximately one ⁇ g of mRNA was used to program in vitro translation in 25 ⁇ L volume reactions using a nuclease treated rabbit reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's protocol.
  • Proteins were synthesized in a nuclease treated rabbit reticulocyte lysate in the presence of 35 S-Met and then analyzed by SDS-PAGE (12.5% polyacrylamide) and autoradiography. However, since TMV CP contains no methionine residues, 3 H-Leu was used when the TMV CP ORF was translated in vitro. Proteins translated in vitro were analyzed by autoradiography following SDS-PAGE according to the method of U. Laemmli (Nature, [London] 227:680-685, 1970).
  • pPROl encodes NIa protease activity
  • constructs were engineered in which the CP ORF from tobacco mosaic tobamovirus (TMV) was inserted into the cassette vector provided herein. These constructs are shown schematically in Figure 3 A.
  • processing of the resultant polyprotein was due to recognition and cleavage of the specific heptapeptides by the NIa protease and not to non-specific degradation, two additional controls were designed.
  • the C-terminal NIa protease domain was removed with a frameshift mutation at the unique BamHI site, resulting in PPROIT ⁇ N ( Figure 3 A). In this construct, processing is not expected despite the presence of the naturally occurring cleavage sequence.
  • the two target heptapeptides were mutated to include a Gin to His change at the -1 position. This mutation at the cleavage site has been previously shown to inhibit the specific processing by NIa in TEV (Dougherty, et al, 1988, supra; Dougherty, et al, 1989, supra).
  • the resulting mutant cassette vector was named pPRO4 and the corresponding pPRO4.NT and pPRO4.TN were also constructed as shown in Figure 3A.
  • the CP produced from pPROl.TN was slightly larger than that produced from pPROl.NT, in accordance with the numbers of amino acid residues added when the cDNA was cloned at the Sma I site versus the Stu I site (see Figure IC).
  • the major proteins resulting from constructs pPRO4.NT and pPRO4.TN migrated at positions corresponding to the size of the precursor polypeptide containing NIa plus TMV CP (68 kDa).
  • the protease domain from NIa was absent (JJPROI .T ⁇ N) a single protein of about 28 kDa, corresponding to the truncated protein, was detected.
  • pPROl was further tested with the introduction of coding sequences for two different heterologous proteins into the two insertion sites.
  • Figure 4 A shows the resulting constructs pPROl.SNT and pPROl.TNS.
  • Figure 4B in vitro transcription and translation of these two constructs gave the predicted patterns of labeled proteins, resulting in the accumulation of proteins with the expected sizes of the NIa (49 kDa), SMV CP (around 30 kDa) and TMV CP (around 18 kDa).
  • the coat proteins inserted at the Sma I site of pPRO 1 gave slightly larger mature proteins than those inserted at the Stu I site due to incorporation of extra peptides as described in Figure IC.
  • the more rapidly migrating proteins (predicted to be the TMV CP) co-migrated with proteins recovered following immunoprecipitation with anti-TMV CP antibody as in Example 2 above.
  • FIG. 5 A Another construct, pPROl.SNG shown in Figure 5 A, consisted of the SMV CP positioned at the Sma I insertion site of pPROl and the open reading frame encoding the ⁇ -glucuronidase activity (GUS) at the Stu I insertion site of pPROl.
  • GUS ⁇ -glucuronidase activity
  • Figure 5B following in vitro translation in the presence of 35 S-Met, the expected profile of mature proteins was generated.
  • the polypeptide synthesized upon translation of this construct has a predicted size of about 149 kDa, and is the largest that has been tested with the pPRO 1 expression cassette. In this particular case, a high molecular weight band corresponding to a polypeptide of approximately 110 kDa was present in relatively low amounts. This protein probably corresponds to a fusion of the NIa and GUS peptides, implying that processing was not complete.
  • Sequence ID No. 1 is an amino acid sequence for the consensus heptapeptide cleavage sequences that are cleaved by the NIa from TEV.
  • Sequence ID No. 2 is an amino acid sequence for the consensus heptapeptide cleavage sequences that are cleaved by the NIa from TEV.
  • Sequence ID No. 3 is an amino acid sequence for a self-recognized heptapeptide cleavage sequences at the N terminus of NIa in TEV.
  • Sequence ID No. 4 is an amino acid sequence for a self-recognized heptapeptide cleavage sequence C terminus of NIa in TEV.
  • Sequence ID No. 5 is a nucleotide sequence for PRO1 ( Figure IB).
  • Sequence ID No 6 is an amino acid sequence for PRO1 ( Figure IB).
  • Sequence ID No. 7 is a nucleotide sequence for a primer (TEVNIA.N) for amplification and cloning of cDNA encoding the nuclear inclusion a protein of tobacco etch potyvirus.
  • Sequence ID No 8 is a nucleotide sequence for a primer (TEVNIA.C) for amplification and cloning of cDNA encoding the nuclear inclusion a protein of tobacco etch potyvirus.
  • Sequence ID No. 9 is a nucleotide sequence for a primer (TEVNTR.5) for amplification and cloning of the 5' untranslated region of tobacco etch potyvirus.
  • Sequence ID No 10 is a nucleotide sequence for a primer (TEVNTR.3) for amplification and cloning of the 5' untranslated region of tobacco etch potyvirus.
  • Sequence ID No. 11 is a nucleotide sequence for a primer (TEVNIA.N2) for amplification and cloning of cDNA encoding the nuclear inclusion protein of tobacco etch potyvirus.
  • Sequence ID No 12 is a nucleotide sequence for a primer (TEVNIA.C3) for amplification and cloning of cDNA encoding the nuclear inclusion protein of tobacco etch potyvirus.
  • Sequence ID No. 13 is a nucleotide sequence for a primer (TMVCP.51) for amplification and cloning of cDNA encoding the tobacco mosaic virus coat protein.
  • Sequence ID No 14 is a nucleotide sequence for a primer (TMVCP.31 ) for amplification and cloning of cDNA encoding the tobacco mosaic virus coat protein.
  • Sequence ID No. 15 is a nucleotide sequence for a primer (SMVCP.N1) for amplification and cloning of cDNA encoding the soybean mosaic virus coat protein.
  • Sequence ID No. 16 is a nucleotide sequence for a primer (SMVCP.C2) for amplification and cloning of cDNA encoding the soybean mosaic virus coat protein.
  • Sequence ID No. 17 is a nucleotide sequence for a primer (GUS.N2) for amplification and cloning of cDNA encoding ⁇ -glucuronidase.
  • Sequence ID No. 18 is a nucleotide sequence for a primer (GUS.C1) for amplification and cloning of cDNA encoding ⁇ -glucuronidase.
  • MOLECULE TYPE DNA (genomic)
  • AATCAAGCAT TCTACTTCTA TTGCAGCAAT TTAAATCATT TCTTTTAAAG CAAAAGCAAT 12
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • IMMEDIATE SOURCE
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne une cassette permettant l'expression simultanée d'au moins deux peptides hétérogènes en quantités équimolaires et sur la base de la protéase d'inclusion nucléaire (NIa) issue du potyvirus etch du tabac. Les peptides hétérogènes sont traduits et incorporés dans un polypeptide qui comporte également la protéase, laquelle assure une libération post-traductionnelle des protéines hétérologues par réaction autoprotéolytique.
PCT/US1995/001495 1994-02-03 1995-02-03 Cassette pour accumuler de multiples proteines par synthese d'un polypeptide a traitement auto-proteolytique WO1995021249A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0700999A2 (fr) * 1994-07-13 1996-03-13 Sankyo Company Limited Système d'expression utilisant des proteines de fusion autolysés et un nouveau polypeptide
WO2000011175A1 (fr) * 1998-08-18 2000-03-02 Syngenta Limited Methode genetique d'expression de polyproteines dans des plantes
WO2001059091A2 (fr) * 2000-02-11 2001-08-16 Metabolix, Inc. Constructions a expression de genes mutiples contenant des inteines modifiees
ES2161130A1 (es) * 1999-04-07 2001-11-16 Consejo Superior Investigacion Dna recombinante derivado del virus de la sharka y vector de expresion de proteinas heterologas basado en dicho dna recombinante.
WO2002061100A1 (fr) * 2001-01-30 2002-08-08 Wisconsin Alumni Research Foundation Expression de proteines multiples dans des plantes transgeniques
EP1629095A2 (fr) * 2003-06-03 2006-03-01 Cell Genesys, Inc. Compositions et procedes permettant l'expression accrue d'immunoglobulines a partir d'un seul vecteur utilisant un site de clivage peptidique
EP1636360A1 (fr) * 2003-06-03 2006-03-22 Cell Genesys, Inc. Compositions et methodes pour une expression amelioree de polypeptides recombinants a partir d'un vecteur unique, faisant appel a un site de clivage peptidique
EP2468881A2 (fr) 2005-07-21 2012-06-27 Abbott Laboratories Expression de gènes multiples incluant des constructions molles avec des polyprotéines, des pro-polyprotéines et protéolyse
US8945876B2 (en) 2011-11-23 2015-02-03 University Of Hawaii Auto-processing domains for polypeptide expression

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162601A (en) * 1989-11-22 1992-11-10 The Upjohn Company Plant potyvirus expression vector with a gene for protease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162601A (en) * 1989-11-22 1992-11-10 The Upjohn Company Plant potyvirus expression vector with a gene for protease

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6743611B2 (en) 1994-07-13 2004-06-01 Sankyo Company, Limited Expression systems utilizing autolyzing fusion proteins and a novel reducing polypeptide
EP0700999A3 (fr) * 1994-07-13 1997-04-16 Sankyo Co Système d'expression utilisant des proteines de fusion autolysés et un nouveau polypeptide
US5955072A (en) * 1994-07-13 1999-09-21 Sankyo Company, Limited Expression systems utilizing autolyzing fusion proteins and a reducing polypeptide
EP0700999A2 (fr) * 1994-07-13 1996-03-13 Sankyo Company Limited Système d'expression utilisant des proteines de fusion autolysés et un nouveau polypeptide
US6307038B1 (en) 1994-07-13 2001-10-23 Sankyo Company, Limited Expression systems utilizing autolyzing fusion proteins and a novel reducing polypeptide
WO2000011175A1 (fr) * 1998-08-18 2000-03-02 Syngenta Limited Methode genetique d'expression de polyproteines dans des plantes
ES2161130A1 (es) * 1999-04-07 2001-11-16 Consejo Superior Investigacion Dna recombinante derivado del virus de la sharka y vector de expresion de proteinas heterologas basado en dicho dna recombinante.
US7741530B2 (en) 2000-02-11 2010-06-22 Metabolix, Inc. Multi-gene expression constructs containing modified inteins
EP2159288A2 (fr) * 2000-02-11 2010-03-03 Metabolix, Inc. Construction pour expression multigénique portante des intéines modifiées
WO2001059091A3 (fr) * 2000-02-11 2001-12-20 Metabolix Inc Constructions a expression de genes mutiples contenant des inteines modifiees
EP2159287A3 (fr) * 2000-02-11 2014-02-12 Metabolix, Inc. Construction pour expression multigénique portant des intéines modifiées.
EP2159288A3 (fr) * 2000-02-11 2014-02-12 Metabolix, Inc. Construction pour expression multigénique portante des intéines modifiées
US7026526B2 (en) 2000-02-11 2006-04-11 Metabolix, Inc. Multi-gene expression constructs containing modified inteins
US8293971B2 (en) 2000-02-11 2012-10-23 Metabolix, Inc. Multi-gene expression constructs containing modified inteins
WO2001059091A2 (fr) * 2000-02-11 2001-08-16 Metabolix, Inc. Constructions a expression de genes mutiples contenant des inteines modifiees
EP2159287A2 (fr) * 2000-02-11 2010-03-03 Metabolix, Inc. Construction pour expression multigénique portant des intéines modifiées.
WO2002061100A1 (fr) * 2001-01-30 2002-08-08 Wisconsin Alumni Research Foundation Expression de proteines multiples dans des plantes transgeniques
EP1629095A4 (fr) * 2003-06-03 2006-06-21 Cell Genesys Inc Compositions et procedes permettant l'expression accrue d'immunoglobulines a partir d'un seul vecteur utilisant un site de clivage peptidique
US7662623B2 (en) 2003-06-03 2010-02-16 Biosante Pharmaceuticals, Inc. Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site
US7498024B2 (en) 2003-06-03 2009-03-03 Cell Genesys, Inc. Compositions and methods for enhanced expression of immunoglobulins from a single vector using a peptide cleavage site
US7709224B2 (en) 2003-06-03 2010-05-04 Biosante Pharmaceuticals, Inc. Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site
US7485291B2 (en) 2003-06-03 2009-02-03 Cell Genesys, Inc. Compositions and methods for generating multiple polypeptides from a single vector using a virus derived peptide cleavage site, and uses thereof
EP1636360A4 (fr) * 2003-06-03 2006-11-08 Cell Genesys Inc Compositions et methodes pour une expression amelioree de polypeptides recombinants a partir d'un vecteur unique, faisant appel a un site de clivage peptidique
EP1636360A1 (fr) * 2003-06-03 2006-03-22 Cell Genesys, Inc. Compositions et methodes pour une expression amelioree de polypeptides recombinants a partir d'un vecteur unique, faisant appel a un site de clivage peptidique
EP1629095A2 (fr) * 2003-06-03 2006-03-01 Cell Genesys, Inc. Compositions et procedes permettant l'expression accrue d'immunoglobulines a partir d'un seul vecteur utilisant un site de clivage peptidique
EP2468881A2 (fr) 2005-07-21 2012-06-27 Abbott Laboratories Expression de gènes multiples incluant des constructions molles avec des polyprotéines, des pro-polyprotéines et protéolyse
EP2468768A2 (fr) 2005-07-21 2012-06-27 Abbott Laboratories Expression de gènes multiples incluant des constructions molles avec des polyprotéines, des pro-polyprotéines et protéolyse
EP2484774A2 (fr) 2005-07-21 2012-08-08 Abbott Laboratories Expression de gènes multiples incluant des constructions molles avec des polyprotéines, des pro-polyprotéines et protéolyse
US8945876B2 (en) 2011-11-23 2015-02-03 University Of Hawaii Auto-processing domains for polypeptide expression

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