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Recombinant expression of multiprotein complexes using polygenes

A multi-gene and gene technology, applied in the field of expressing multi-protein complexes by using multi-gene recombination

Inactive Publication Date: 2009-01-28
ETH ZZURICH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A disadvantage of this method is that two separate vectors must be prepared and two transfections must be performed in order to reconstitute the complete complex

Method used

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  • Recombinant expression of multiprotein complexes using polygenes
  • Recombinant expression of multiprotein complexes using polygenes
  • Recombinant expression of multiprotein complexes using polygenes

Examples

Experimental program
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Effect test

Embodiment 1

[0155] Embodiment 1: Prepare polygene and link in expression vector

[0156] The principle of multiple gene preparation is shown here by utilizing the human TATA-Box binding protein (hTBP) core (hTBPc, a c-terminal fragment of the full-length protein truncated at position 159). The gene encoding hTBPc was amplified by polymerase chain reaction (PCR) using a sense primer that binds to an overhang containing a RsrII restriction site and further encodes an amino acid spacer and tobacco etch The 5' end of the gene of the viral (TEV) cleavage site. The antisense primer binds to the 3' end of the gene and contains a RsrII restriction site. RsrII is a restriction enzyme that produces an asymmetric overhang of 3 nucleotides that cannot self-ligate, therefore, the restriction product is asymmetric and the ligation produces a directional product. The PCR product was digested with RsrII and purified. figure 1 The DNA (SEQ ID NO: 1 ) and deduced amino acid sequence (SEQ ID NO: 2) of...

Embodiment 2

[0158] Example 2: Construction of a baculovirus containing multiple genes encoding human universal transcription factor subunits carrier

[0159] Formation of polygenes inserted into the transfer vector pUCDMA expressed by baculovirus (see WO2005 / 085456A1 (PCT / EP2004 / 013381)) encoding polyproteins including human TBP-associated factors hTAF1 and hTAF2 in addition to hTBPc , the genes in the polygene are separated by sequences encoding amino acid spacers and TEV protease sites. Figure 9 A schematic diagram of the resulting construct pUCDMCSTAF1TBPcTAF2 is shown. The nucleotide sequence of this construct is shown in Figure 10 Medium (SEQ ID NO: 4). Another one that forms a polygene encoding a polyprotein including TEV protease and human TBP-associated factors hTAF6 and hTAF9 inserted into the transfer vector pFBDM expressed by baculovirus (see WO2005 / 085456A1 (PCT / EP2004 / 013381)) Constructs, the genes in the polygene are separated by sequences encoding amino acid spacer...

Embodiment 3

[0160] Example 3: Preparation of bacmid constructs, insect cell infection and protein expression

[0161] In order to construct bacmid constructs containing the above two polygenes, the constructs pUCDMCSTAF1TBPcTAF2 (pUCDM derivative) and pFDDO[HisTEVTAF6TAF9]his (pFBDM derivative) were each introduced in WO2005 / 085456A1 (PCT / EP2004 / 013381)) Example 5 (for pUCDMCSTAF1TBPcTAF2; Cre-lox site-specific recombination) and the DH10MultiBac described in Example 6 (for pFDDO[HisTEVTAF6TAF9]his; Tn7 transposition) Cre in cells. If desired, the DH10MultiBac described in WO2005 / 085456A1 (PCT / EP2004 / 013381) Cre One-step transposition / cre-lox site-specific recombination on cells. Preparation of bacmids, insect infection and protein expression can be carried out according to established methods (see, for example, O'Reilly et al. (1994) "Baculovirus expression vector. Alaboratory manual" Oxford University Press, New York-Oxford; "Bac -toBac TM Baculovirus Expression Systems Manual" I...

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Abstract

The present invention relates to a recombinant polynucleotide encoding a polygene coding for at least three polypeptides wherein at least one of the genes constituting the polygene is of non-viral origin, at least two of the polypeptides encoded by the genes constituting the polygene are each capable of at least transiently interacting with at least one other polypeptide encoded by a gene of said polygene, and the genes constituting the polygene are each connected to one another by a sequence coding for at least one protease cleavage site. The present invention also relates to polyproteins encoded by the polygene. Further embodiments of the present invention are a vector containing the recombinant polypeptide, a host cell containing the recombinant polypeptide and / or the vector and a non-human transgenic animal transformed with the recombinant polypeptide and / or the vector. The present invention also relates to methods for the production of the polynucleotide and for the manufacture of multiprotein complexes. The embodiments of the present invention are particularly useful in gene therapy, drug candidate screening, vaccine production and crystallisation of multiprotein complexes for structural investigations.

Description

technical field [0001] The present invention relates to recombinant polynucleotides encoding at least two polygenes each encoding at least three biologically active polypeptides, each polygene being in a single open reading frame (ORF), wherein the genes constituting the polygene encode at least two polypeptides are of non-viral origin, each of the at least two polypeptides encoded by the genes comprising the polygene is capable of at least transiently interacting with at least one other polypeptide encoded by the genes comprising the polygene, and constituting The genes of each polygene are linked to each other by a sequence encoding at least one protease cleavage site and / or by a sequence encoding at least one self-cleaving peptide. Other embodiments of the present invention are vectors containing recombinant polynucleotides, host cells containing recombinant polynucleotides and / or vectors, and transgenic animals other than humans transformed with the recombinant polynucleot...

Claims

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Application Information

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IPC IPC(8): C12N15/79A01K67/027C12N15/866A61K48/00C12N15/10
CPCC07K14/4702C07K2319/50C07K2319/21C12N2799/026C12N15/85A61P43/00C12N15/10C12N15/866C12N15/79A61K48/00C07K2319/80C12N15/86C12N2710/14043
Inventor T·里克蒙德I·伯格
Owner ETH ZZURICH
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